Maria Veiga-Da-Cunha
email: veigadacunha@bchm.ucl.ac.

Patrick Jacquemin

Ghislain Delpierre

Catherine Godfraind

Ivan Théate

Didier Vertommen

Frédéric Clotman

Frédéric Lemaigre

Olivier Devuyst

Emile Van Schaftingen

Increased protein glycation in fructosamine-3-kinase-deficient mice

Keywords: Intracellular glycation, Fructosamine-3-kinase inactivation, Protein repair, Fructoselysine, Diabetic complications

INTRODUCTION

Glucose spontaneously reacts with amines to form Schiff bases, which slowly undergo an Amadori rearrangement to become more stable, fructose-like compounds known as fructosamines [START_REF] Hodge | The Amadori Rearrangement[END_REF][START_REF] Baynes | The Amadori product on protein: structure and reactions[END_REF]. Originally identified in vitro, the formation of fructosamines (known as early glycation), takes place also in living organisms. Many intracellular (e.g., hemoglobin) and extracellular proteins (e.g. collagen, serum albumin) are known to bear fructosamines on lysine residues or on their aminoterminal amine. Unlike enzymecatalyzed post-translational modifications, protein glycation is largely substoichiometric. Fructosamine formation is proportional to glucose concentration and glycation is therefore enhanced in diabetes [START_REF] Baynes | The Amadori product on protein: structure and reactions[END_REF][START_REF] Brownlee | Glycation and diabetic complications[END_REF].

Until recently, it was thought that the formation of fructosamines was an irreversible process, which is unavoidably followed by their slow conversion to a series of compounds known as advanced glycation end-products (AGEs). The latter are thought to participate in the development of diabetic complications [START_REF] Baynes | The Amadori product on protein: structure and reactions[END_REF][START_REF] Brownlee | Nonenzymatic glycosylation and the pathogenesis of diabetic complications[END_REF]. However, the recent identification of fructosamine-3-kinase (FN3K), an enzyme able to convert free and protein-bound fructose--lysine residues [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF][START_REF] Szwergold | Human fructosamine-3-kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo[END_REF] to unstable fructosamine 3-phosphates, opened the perspective that fructosamines can be physiologically removed from proteins. The finding that the presence of a competitive inhibitor of FN3K increases the accumulation of glycated hemoglobin in erythrocytes incubated with 200 mM glucose [START_REF] Delpierre | Fructosamine-3-kinase is involved in an intracellular deglycation pathway[END_REF] indicates that at least under these non-physiological conditions, FN3K may act to repair hemoglobin. However, it still needs to be proven that this mechanism takes place in vivo, at physiological concentrations of glucose, and that it also applies to other cell types than erythrocytes. Furthermore, in vitro, FN3K phosphorylates free fructose--lysine with high affinity [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF], and it could also be involved in the metabolism of this compound. To investigate these points, we have carried out the inactivation of the FN3K gene in mice.

EXPERIMENTAL Materials

Reagents were of analytical grade. Radiochemicals were from GE Healthcare (Roosendaal, The Netherlands). AG 1X-8 (200-400 mesh) and AG 50W-X4 (100-200 mesh) resins were from BioRad Laboratories (Hercules, CA, USA). Auxiliary enzymes and enzyme substrates were from Roche Molecular Biochemicals (Mannheim, Germany) and Sigma-Aldrich Chemical (St. Louis, MO, USA).

Recombinant E. coli fructoselysine-6-kinase [START_REF] Wiame | Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli[END_REF] was tagged with 6-His residues at the N-terminus and over-expressed in E. coli (24 h at 19°C). It was purified to near homogeneity by Ni ++ affinity chromatography after elution with a step-wise imidazole gradient from a HisTrap column (GE Healthcare, Roosendaal, The Netherlands). The active fractions were concentrated, desalted by gel filtration, enriched with 10% glycerol and stored at -80°C. The purified preparation (85 U/ml; 2.4 mg/ml) was clear from contaminating phosphatase activities.

Animals

All mice were raised under standard husbandry conditions and were housed in colony cages with a 12 hour light/dark cycle. They had free access to water and food, unless otherwise specified. Mice used in this study were the offspring of fourth and fifth generation heterozygous (FN3K +/-) mice. The experiments were performed with the approval of the ethical committee of the host institution.

Construction of the targeting vector and generation of knockout mice

The targeting vector was obtained by directly subcloning fragments of the FN3K gene from the female mouse 129S6/SvEvTac RPCI-21 PAC Library (CHORI-BACPAC Resources, Bruce Lyon Memorial Res.

Lab, Oakland, CA 94409, USA) and the neomycin resistance gene, with a PGK promoter and polyA signal, from pPNT (9) in the pSKT-NLSLacZ vector (Fig. 1A). The heterologous genes were flanked by 5.5 kb of sequence upstream of the FN3K initiator ATG codon and 2.8 kb of sequence downstream of the second exon. This construct was electroporated in mouse R1 embryonic stem cells (a gift from A. Nagy, Leuven). PCR analysis of 300 neomycin-resistant clones indicated that 12 of them had undergone a sitespecific recombination event. Southern blot analysis (Fig. 1B) and karyotyping of 5 of them allowed the selection of an appropriate ES clone, which was aggregated to CD1 morulae and the resulting blastocysts were transferred into pseudo-pregnant mice. Twenty-six chimeric mice were born, 23 of which were males. Six of these were mated with CD1 females and 5 of them transmitted the deletion in the FN3K gene. Heterozygous offspring was crossed to generate knockout progeny, which was therefore in a mixed CD1/129 background. The mice were genotyped by PCR with primers corresponding to DNA sequences in exon 2 and in the neo cassette: exon 2-S 5'-GATGTTTGAGGGAGAGATGGC-3' and exon 2-AS 5'-GGCTCTTCATCTTCAAGTGCT-3'; neo-S 5'-TCCCCTCAGAAGAACTCGTC-3' and neo-AS 5'-AGCACGTACTCGGATGGAAG-3'.

FN3K activity and total glycated hemoglobin (GlcHb)

For the assay of mouse FN3K activity, the conversion of [ 14 C]fructose--lysine [labeled on its deoxyfructose moiety [START_REF] Delpierre | Fructosamine-3-kinase is involved in an intracellular deglycation pathway[END_REF]], to [ 14 C]fructoselysine-3-phosphate was measured in cell extracts (erythrocytes) or after partial purification on Blue Sepharose (kidney and brain) [START_REF] Delplanque | Tissue distribution and evolution of fructosamine 3-kinase and fructosamine 3-kinase-related protein[END_REF]. Glycated hemoglobin (GlcHb) was measured using phenylboronate affinity chromatography with the "Helena Glyco-Tek Affinity Column" kit (Helena BioSciences Europe, Sunderland, UK), according to the manufacturer's protocol except that the red blood cells were left in the lysis solution for 30 min before loading onto the columns. According to Abraham et al. (11), the Schiff's bases are not retained on this type of column.

Detection of fructosamines in red blood cell extracts by phosphorylation with fructoselysine-6kinase after separation by SDS-PAGE

Cell extracts were submitted to SDS-PAGE in a 12% (w/v) polyacrylamide gel. The proteins were electrophoretically transferred onto a HybondTM-C Extra nitrocellulose membrane (GE Healthcare, Roosendaal, The Netherlands) which was washed successively with 20 mM Hepes, pH 7.1 (3 x 10 min) and blocked by incubation with 5% non-fat dry milk in the same buffer (60 min at 30°C). The membrane was then incubated for 90 min at 30°C with 0.7 U/ml fructoselysine-6-kinase, 20 mM Hepes, pH 7.1, 1 mM MgCl 2 , 25 mM KCl, 20 M ATP-Mg and 6.5 x 10 6 c.p.m./ml [ -32 P]ATP. The phosphorylation was stopped and excess ATP eliminated by washing at 30°C (3 x 5 min) in 20 mM Hepes, pH 7.1 and 1 mM EDTA. The membrane was dried and exposed to a PhosphoImager screen (Molecular Dynamics, Sunnyvale, CA, USA) for detection of radioactivity. The identity of the proteins corresponding to the increase in phosphorylation observed in the FN3K -/-mice was confirmed by mass Spectrometry analysis of the corresponding bands in the SDS-PAGE gels.

Measurement of protein-bound fructoselysine and plasma fructosamines

To measure protein-bound fructoselysine, tissue extracts were extensively dialyzed against water to eliminate free fructose--lysine, and protein (150 g) was then submitted to digestion by pepsin, pronase E, aminopeptidase and prolidase, as described in [START_REF] Ahmed | Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to N--carboxymethyl-lysine and N--(1-carboxyethyl)lysine-modified albumin[END_REF]. Digestion was stopped by extraction with perchloric acid, and free fructose--lysine was measured in the neutralized extract using E. coli fructoselysine-6kinase and [ -32 P]ATP [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF].

The concentration of extracellular fructosamines was measured using 100 l of plasma isolated from mouse blood treated with EDTA. We used a colorimetric assay [START_REF] Baker | Use of protein-based standards in automated colorimetric determinations of fructosamine in serum[END_REF] that relies on the ability of fructosamines to act as reducing agents in alkaline solutions. 1-deoxy-morpholinofructose was chosen as the standard fructosamine.

Assay of glycated enzymes

The extent of glycation of intracellular proteins in various tissues was determined by phenylboronate affinity chromatography using the "Helena Glyco-Tek Affinity Column" kit (Helena BioSciences Europe, Sunderland, UK) in hemolysates prepared from whole blood and in the supernatants of a 1/4 tissue extracts made in 25 mM Hepes, 1 mM DTT, 1 g/ml leupeptine and antipaine. 15 l of whole blood [approx. 2 mg protein and 1.4 U of triose-phosphate isomerase (TPI), or 0.14 U of phosphoglucose isomerase (PGI) and 3-phosphoglycerate kinase (3-PGK) were added to 85 l of the lysis buffer supplied with the kit and immediately applied onto the phenylboronate columns. For the tissues the volume of extract added to the lysis buffer (100 l final volume) varied and corresponded to approx. 3U of TPI, and 0.4 U of PGI and 3-PGK. In order to standardize the amount of protein loaded onto the phenylboronate columns (2 mg), BSA was added to the diluted extract.

After loading the columns at room temperature, the samples were left for 10 min before washing with 0.5 ml followed by 4 ml of solution A (50 mM MgCl 2 , 100 mM glycine, pH 8.1 to 8.6, supplied with the kit)-fraction 1. The retained glycated proteins were eluted with 3 ml of 20 mM Hepes, pH 8.5, 350 mM sorbitol and 0,01% sodium azide (fraction 2). This solution was preferred to solution B (provided by the manufacturer), since the latter was found to cause inhibition of the enzymatic activity. In order to preserve the enzymatic activity we added 0.5 mg/ml BSA to fraction 2, immediately after elution from the column. Fractions 1 and 2 were kept on ice and their enzymatic activities measured spectrophotometrically at 30°C and used to compute the proportion of enzyme that was retained on the column. Some of the enzymatic activities detected in fractions 1 and 2 did not stand freezing and thawing and had to be measured soon after elution from the column. When working as described, we recovered on average 100% (TPI, PGI) and 70% (3-PGK) of the activities in the extracts loaded onto the columns. TPI was measured in 25 mM Hepes, pH 7.1, 25 mM KCl, 1 mM MgCl 2 , 0.17 mM NADH+H + , 1 U/ml of desalted rabbit muscle glycerol 3-phosphate dehydrogenase and 40 l or 15 l of fraction 1 or 450 l or 100 l of fraction 2 for red blood cells and other tissues, respectively. The reaction was started by addition of 1 mM glyceraldehyde 3-phosphate. PGI was measured in 25 mM Hepes, pH 7.1, 2 mM MgCl 2 , 1 mM DTT, 0.25 mM NADP + +H + , 1 U/ml of desalted yeast glucose 6-phosphate dehydrogenase and 50 l of fraction 1 or 450 l of fraction 2. The reaction was started by addition of 2 mM fructose 6phosphate. 3-PGK was measured in 85 mM Tri-ethanolamine buffer pH 7.6, 2 mM EDTA, 1.5 mM MgSO 4 , 7 mM 3-phosphoglyceric acid, 1 U/ml of glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle and 50 l of fraction 1 or 450 l of fraction 2. The reaction was started by addition of 1 mM ATP-Mg.

Protein concentration in tissue samples was measured using -globulin as a standard [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding[END_REF].

Assay of free fructose--lysine

Free fructose--lysine was assayed in neutralized perchloric acid extracts from red blood cells, plasma, tissues and urine of FN3K +/+ and FN3K -/-mice. Freshly collected mouse blood treated with EDTA was centrifuged (10 min at 800 x g at 4°C) to recover the plasma. Red blood cells were then washed 3 times with 0.15 M NaCl and the pellet diluted with 1 vol. of H 2 O. Perchloric acid extracts (5%) were made by addition of 70% HClO 4 to the diluted red blood cells or directly to the plasma or urine. For the tissues, the Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060684 frozen material was directly homogenized in 2 vol. of 10% HClO 4 . After centrifugation, the supernatant was neutralized with 3M KHCO 3 and with the exception of urine, diluted with 2 vol. of H 2 O and applied onto a AG 1-X8 column (Cl -form, 1 ml) equilibrated in H 2 O in order to eliminate ATP. The flow-through and the wash fractions (4 ml) containing fructoselysine were collected, lyophilised and rediluted (10 mM Hepes, pH 7.1) in the initial sample volume. Free fructose--lysine was determined using a radiochemical assay where fructose--lysine is phosphorylated to fructoselysine 6-phosphate in the presence of 0.5 U of The fructose--lysine in the neutralized perchloric acid extracts of urine was assayed spectrophotometrically using fructoselysine-6-kinase, pyruvate kinase and lactate dehydrogenase in an ATP-coupled assay previously described [START_REF] Wiame | Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli[END_REF].

The concentration of fructose--lysine measured in urine and red blood cells is normalized to the concentration of creatinine [START_REF] Jaffé | Uber den niederschlag, welchen pikrinsaure in normalen harn erzeugt und uber eine neue reaction des kreatininis[END_REF] or hemoglobin [START_REF] Drabkin | Spectrophotometric studies. Preparations from washed blood cells, nitric oxide hemoglobin and sulfhemoglobin[END_REF] in the respective samples. Fructose--lysine in tissues is calculated in relation to the weight of the samples.

Histological examinations

All tissue samples taken from FN3K +/+ and FN3K -/-mice were immediately fixed in formalin and routinely processed for paraffin embedding. Tissue sections (5 m) were stained (hematoxylin-eosin, periodic acid Schiff and Masson's blue trichrome stain) and examined by two different pathologists (C.G. and I.T.).

RESULTS

Inactivation of the FN3K gene

The construct that was used to inactivate the FN3K gene through homologous recombination (Fig. 1A) was designed so as to replace the first exon, the first intron and the second exon of the mouse FN3K gene by an in-frame bacterial -galactosidase gene and a neomycin-resistance gene. FN3K -/-mice were viable and the genotyping of 736 pups born from heterozygous matings showed that their distribution was as expected for unbiased Mendelian inheritance. There was no difference in fertility or in litter size for FN3K -/-mice. RT-PCR analysis of RNA from various tissues indicated the absence of FN3K mRNA in FN3K -/-mice (Fig. 1C). Furthermore, FN3K activity was undetectable in erythrocytes, brain and kidney extracts (Fig. 1D) of FN3K -/-mice and, in erythrocyte extracts from FN3K +/-mice it amounted to 50 % of the activity measured for wild type mice.

Since the -galactosidase transgene was placed in frame with the initiator ATG codon of the FN3K gene, we also tried to detect the presence of -galactosidase in tissues from FN3K -/-mice by activity staining and RT-PCR analysis. However, activity staining did not disclose the presence of ßgalactosidase and RT-PCR revealed only very low amounts of its mRNA in adult brain, liver or kidney, which are tissues where FN3K is normally expressed [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF][START_REF] Delplanque | Tissue distribution and evolution of fructosamine 3-kinase and fructosamine 3-kinase-related protein[END_REF]. These data indicated that the -galactosidase transgene did not recapitulate the normal expression of the FN3K gene, suggesting that the region from exon 1 to exon 2 of the FN3K gene contains important regulatory elements.

General characterization of the FN3K -/-mice

For both males and females, the ablation of the FN3K gene did not affect growth, as indicated by the fact that there was no difference in body weight between FN3K -/-and control mice at various ages (from 3 weeks to 10 months). For instance at 15 weeks, the body weight (in g) was 34.8 ± 1.2 and 34.2 ± 1.0 for male FN3K +/+ and FN3K -/-mice, respectively and 27.1 ± 1.0 and 26.9 ± 0.8 for females. No difference in the blood glucose concentration was observed for overnight starved mice aged 1, 2, 4 and 6 months (n=4 for each age and sex). Furthermore, the concentration in extracellular plasma fructosamines was not different in 18-month old FN3K -/-(1.59 ± 0.16 mM; n=8) and in wild-type (1.61 ± 0.10 mM; n=8) mice.

No glucosuria or increased urinary albumin excretion was observed (not shown) in FN3K -/-mice (males, aged 2 to 12 months) and their plasma urea or creatinine concentrations were normal. The FN3K -/-mice looked healthy and there was no apparent difference in life span ( 2 years) compared to control mice.

Histological examinations of tissues from FN3K -/-mice (brain, cerebellum, eyes, thymus, kidney, liver, heart, skeletal muscle, spleen, bone marrow, pancreas, stomach, intestine, testis, ovary, uterus, bone marrow) performed at birth, 2, 4, 12 and 18 months did not disclose any particular finding compared to wild type mice of the same age.

Effect of FN3K deficiency on hemoglobin glycation

The impact of FN3K ablation on protein glycation was determined by measuring GlcHb by affinity chromatography on phenylboronate columns in blood from overnight-starved mice aged 1 to 6 months. An Copyright 2006 Biochemical Society approximate 2.5-fold increase in the amount of glycated hemoglobin was observed in FN3K -/-mice, whereas FN3K +/-mice had a GlcHb level similar to that observed with control mice (Fig. 2).

To confirm that the increased glycation observed in FN3K -/-mice corresponded to an increase in protein-bound fructosamines, erythrocyte proteins were submitted to SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with [ - 32 P]ATP and E. coli fructoselysine-6-kinase. This enzyme acts best on free fructose--lysine, but also, more slowly, on protein-bound fructoselysine [START_REF] Wiame | Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli[END_REF]. As shown in Fig. 3, several fructosamine-containing bands were detected in this way and these were significantly more abundant in erythrocytes of FN3K -/-versus FN3K +/+ mice. Mass spectrometry analysis of tryptic peptides indicated that the lowest, most abundant band corresponded to hemoglobin monomers, the intermediate one, also to hemoglobin (probably dimers), and the two upper bands, to carbonic dehydratase II and hemoglobin (possibly tetramers). To compare the extent of glycation at various sites in hemoglobin from FN3K -/-and FN3K +/+ mice, we used a method [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF] in which the fructosamine residues are tagged with [ 32 P] by incubation with [ -32 P]ATP and FN3K. After reduction of the fructosamine-3phosphates with borohydride, the protein is digested with trypsin, the resulting peptides are separated by HPLC and the radioactivity is counted in the fractions. Typical HPLC profiles, illustrated in Fig. 4, show that the incorporated radioactivity was globally higher in hemoglobin from FN3K -/-mice than from wild type mice, consistent with a higher degree of glycation in the FN3K deficient mice. Remarkably, some of the peaks found with hemoglobin from the FN3K -/-mice were almost absent from the profiles corresponding to the wild type mice whereas other peaks were much less affected. These data indicated that in vivo deglycation takes place with efficiencies that differ largely from site to site.

Glycation of proteins in tissues

Due to the wide tissue distribution of FN3K, we anticipated that FN3K ablation would cause an increase in the level of glycated proteins in many tissues. To investigate this point, we first determined the level of protein-bound fructoselysine with a specific enzymatic fructose--lysine assay after extensive proteolytic digestion of total brain, kidney, liver, and erythrocytes protein from 1, 2 and 4 month-old mice. In the three age groups, protein-bound fructoselysine was consistently higher in erythrocytes and tissues from animals from the same age group. An increase of 20% on average was measured in tissues from FN3K -/- mice compared to wild type mice (brain: 0.16 ± 0.01 vs 0.12 ± 0.01, p= 0.05; kidney: 0.18 ± 0.002 vs 0.16 ± 0.005, p=0.035; liver: 0.17 ± 0.02 vs 0.13 ± 0.01, p=0.2, results expressed as nmol fructose--lysine/mg protein, n=3), whereas it was more than doubled in erythrocytes (FN3K -/-: 0.78 ± 0.22 vs FN3K +/+ : 0.32 ± 0.05 nmol fructoselysine/mg protein, p=0.1, n=3). As FN3K is a cytosolic protein, it was likely that the difference in total (i.e. both intracellular and extracellular) protein-bound fructoselysine values mentioned above underestimated the difference in the glycation level of cytosolic proteins. We therefore measured

Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060684

Copyright 2006 Biochemical Society the glycation extent of 3 cytosolic enzymes, TPI, PGI and 3-PGK by determining their degree of retention on phenylboronate affinity columns. The choice of these enzymes was directed by their high specific activity and their stability in cell extracts. Their glycation was found to be about 2-fold (TPI and 3-PGK) and 1.5-fold higher (PGI) in erythrocyte extracts from FN3K -/-mice than from wild type mice (Fig. 5). TPI and 3-PGK were also found to be more glycated (by 40 to 60%) in liver, kidney, brain and skeletal muscle of FN3K deficient mice (Fig. 6). For PGI the increase in glycation was less important but significant (23% as a mean). FN3K deficiency was not accompanied by any difference in the total activities of these enzymes (not shown).

Effect of FN3K deficiency on tissue and urinary fructose--lysine

As shown in Table 1, free fructose--lysine was present in the plasma of fed mice at a concentration of nearly 3 µM in fed control mice, and this value was not significantly affected by FN3K deficiency. The tissue or cell concentration of free fructose--lysine was lower than 1 nmol/g in brain and heart of FN3K +/+ mice (being there most likely largely contributed by the extracellular compartment), intermediate in skeletal muscle and liver, and the highest in kidneys (probably reflecting contamination by small amounts of urine, which contains about 100-fold higher concentrations of this compound, see below). FN3K deficiency resulted in a marked increase (5 to 13-fold) in the cell or tissue content of fructose--lysine except in kidneys, where the increase in the concentration did not reach statistical significance.

Urine of fed mice contained relatively high concentrations of fructose--lysine (typically of the order of 1 mM) and the urinary excretion of fructose--lysine, as estimated by the fructose-lysine/creatinine ratio did not differ between FN3K +/+ and FN3K -/-mice. The urinary excretion of fructoselysine was, however, 10 to 20-fold lower in 36 h fasted mice, where a significant difference was observed: FN3K -/-excreted 2.5 times more fructose--lysine than FN3K +/+ or FN3K +/-mice (Table 2).

DISCUSSION

Evidence that FN3K acts as a protein deglycating enzyme

The ability of FN3K to convert protein-bound fructosamines to unstable, 3-phospho derivatives suggested that it could act as a protein repair enzyme, responsible for deglycation of intracellular proteins [START_REF] Delpierre | Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase[END_REF][START_REF] Szwergold | Human fructosamine-3-kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo[END_REF][START_REF] Delpierre | Fructosamine-3-kinase is involved in an intracellular deglycation pathway[END_REF]. The present work provides strong evidence for the operation of this mechanism in vivo. Targeted inactivation of the FN3K gene leads to an increase in protein-bound fructosamines, as evidenced by a combination of biochemical analyses, i.e. (1) retention of proteins on phenylboronate affinity columns (which measures the proportion of proteins having cis-diol groups and is therefore not specific for fructosamines), ( 2) measurement of fructose--lysine residues with a specific enzymatic assay, and (3) [ 32 Copyright 2006 Biochemical Society protein-bound fructoselysine with human FN3K or E. coli fructoselysine-6-kinase. This increased glycation affects intracellular (hemoglobin, glycolytic enzymes) but not extracellular proteins, as indicated by the finding that the level of serum fructosamines is the same in FN3K -/-and FN3K +/+ mice. This last observation and the finding that the FN3K -/-mice have a normal blood glucose level, indicates that the difference in protein-bound intracellular fructosamines does not result from a change in glycemia.

Furthermore, the finding that increased glycation affects tissues where glucose entry is carried out by different types of glucose transporters (e.g. Glut1 in erythrocytes, Glut2 in liver and Glut4 in muscle, review in 18) indicates that it is not the result from a change in intracellular glucose concentration consequent to a perturbed regulation of one of the glucose carriers.

The fact that protein-bound fructosamines are present on hemoglobin and on other cytosolic proteins in mice with a normal level of FN3K, indicates that enzymatic deglycation does not proceed to completion. Conceptually, this could be either because FN3K activity is insufficient, i.e. that the rate of glycation largely exceeds the capacity of FN3K, or alternatively, because some of the glycation sites are inaccessible or poorly phosphorylated by FN3K. The findings that the glycated hemoglobin levels in FN3K +/-and FN3K +/+ mice are indistinguishable despite a 2-fold difference in FN3K activity is against the first possibility [as is also the observation that GlcHb as well as HbA1c levels do not differ between humans with an about 4-fold difference in erythrocyte FN3K activity [START_REF] Delpierre | Variability in erythrocyte fructosamine-3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites[END_REF]]. Accordingly the erythrocyte FN3K activity (3 nmol/min/ml of packed cells) largely exceeds the rate of glycation that is calculated to take place at 5 mM glucose ( 0.01 nmol/min/ml packed erythrocyte). This conclusion most likely applies not only to erythrocytes but also to tissues, such as liver and brain, where the FN3K activity (expressed per g tissue) represents at least 10% of the erythrocyte FN3K activity [Fig. 1D and [START_REF] Delplanque | Tissue distribution and evolution of fructosamine 3-kinase and fructosamine 3-kinase-related protein[END_REF]], though we cannot exclude that cell-to-cell differences in the expression of FN3K may make it to be rate-limiting for deglycation in some cell types.

Inaccessibility of FN3K to some glycation sites appears to be the major cause for deglycation being incomplete, as indicated by the large variability in the impact of FN3K ablation on the glycation extent at various sites in hemoglobin. Some of the glycation sites were barely affected by the absence of FN3K, whereas a nearly all-or-none situation was found for others (as e.g. peak 3, Fig. 4), in agreement with results obtained with hemoglobin derived from human erythrocytes incubated with or without a competitive inhibitor of FN3K [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF]. The conclusion from the latter study was that well-accessible fructoselysine residues (e.g. those corresponding to Lys--16, Lys--17, Lys--139 in human hemoglobin) were extensively deglycated in intact erythrocytes (in the absence of the FN3K inhibitor), but that fructosamine removal was negligible at other positions (Val--1, Lys--66), either for accessibility reasons (Lys--66) or because fructosamines bound to alpha amines are much poorer substrates for FN3K than those bound to the side-chain of lysines [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF]. The action of FN3K provides therefore an explanation Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060684 for the long-standing observation that some of the lysines that are preferentially glycated following incubation of purified hemoglobin with glucose are undetectably glycated in intact erythrocytes [START_REF] Shapiro | Sites of nonenzymatic glycosylation of human hemoglobin A[END_REF].

An important question with respect to the monitoring of diabetes is whether variations in FN3K activity lead to changes in the HbA1c value, independent of changes in blood glucose level. The finding that the large variation in FN3K activity (4-fold range) in human erythrocytes does not detectably affect HbA1c levels, suggests that the glycated valine present in human hemoglobin is not a substrate for FN3K in intact cells [START_REF] Delpierre | Variability in erythrocyte fructosamine-3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites[END_REF]. Our attempts to confirm these findings in mice (not shown) were met with difficulties to get a reproducible assay for HbA1c for this species. For instance, mouse HbA1c was poorly separated from other forms of hemoglobin when an HPLC method was used.

Action of FN3K on free fructose--lysine

As FN3K phosphorylates free fructose--lysine with high affinity (Km 10 µM), leading to a compound that spontaneously degrades to lysine, 3-deoxyglucosone and inorganic phosphate [START_REF] Szwergold | Human fructosamine-3-kinase: purification, sequencing, substrate specificity, and evidence of activity in vivo[END_REF], it can participate in the recovery of lysine from its glycated derivative. Our findings indicate that this recovery mechanism applies differently to endogenously-produced and alimentary fructoselysine. That FN3K participates in the metabolism of intracellularly-generated free fructose--lysine is indicated by the finding that the free fructose--lysine concentration was about 5 to 13-fold higher in tissues (to the apparent exception of kidneys, see Table 1) from FN3K -/-mice than from FN3K +/+ mice, whereas no difference was observed for the plasma concentration of this amino acid derivative. Since the increase in intracellular protein glycation amounts to only about 1.5 to 2.5-fold, the 5 to 13-fold increase in tissue free fructose--lysine in FN3K -/-mice is best explained by admitting that FN3K also acts on free fructose--lysine.

The more than 10-fold higher urinary excretion of free fructose--lysine observed in fed compared to starved mice, implies an alimentary origin for most of the urinary fructose--lysine excreted by fed animals. The finding that FN3K deficiency does not affect this excretion in fed mice indicates, therefore, that alimentary fructose--lysine escapes the "salvage" mechanism initiated by FN3K. This agrees with the observation made in mice and other species that parenterally administered fructose--lysine is largely recovered intact in urine after a few hours (21-22, J. Delplanque, unpublished results). It should be noted that orally administered fructose--lysine appears to be largely metabolized by bacteria present in the hindgut of species such as pig, rat and man [START_REF] Mori | Utilization in rats of 14 C-L-lysine labeled casein browned by aminocarbonyl reaction[END_REF][START_REF] Rérat | Nutritional and metabolic consequences of the early Maillard reaction of heat treated milk in the pig. Significance for man[END_REF][START_REF] Lee | Balance experiments on human volunteers with -fructose lysine (FL) and lysinoalanine (LASL)[END_REF], explaining that little alimentary fructose--lysine is recovered in urine in these species. The finding that substantial amounts of fructose--lysine were recovered in urine from fed mice indicates therefore that their intestinal bacterial flora does not comprise fructose--lysine-metabolizing microorganisms. In contrast with fed mice, starved FN3K -/-mice had a significantly higher excretion of fructose-lysine than FN3K +/+ mice. This observation results from the fact that in starved mice, all of the fructose-lysine found in urine comes from the degradation of endogenous proteins (more glycated in FN3K -/-mice than FN3K +/+ mice) and that free fructose--lysine is no longer phosphorylated by FN3K in FN3K -/-mice.

Potential physiological importance of FN3K

The results mentioned above indicate that FN3K can catalyze the repair of proteins in vivo. Protein deglycation by FN3K is therefore a new protein repair mechanism to be recognized, besides the reduction of methionine-sulfoxide residues by peptide methionyl-sulfoxide reductase [START_REF] Grimaud | Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase[END_REF][START_REF] Sun | Repair of oxidized calmodulin by methionine sulfoxide reductase restores ability to activate the plasma membrane Ca-ATPase[END_REF], the repair of isoaspartate residues by isoaspartyl-methyl transferase [START_REF] Clarke | Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair[END_REF], and the isomerisation of cis-proline residues by proline cis-trans isomerase [START_REF] Schiene | Enzymes that catalyze the restructuring of proteins[END_REF]. Mice deficient in either of the first two protein repair mechanisms show a phenotype, quite dramatic in the case of protein L-isoaspartyl/(D-aspartyl) O-methyl transferase (PIMT) deficiency [START_REF] Kim | Deficiency of a proteinrepair enzyme results in the accumulation of altered proteins, retardation of growth and fatal seizures in mice[END_REF][START_REF] Kim | Phenotypic analysis of seizure-prone mice lacking L-isoaspartate (D-aspartate) O-methyltransferase[END_REF] and much milder and dependent on the O 2 concentration in the case of methionine sulfoxide reductase (MsrA) deficiency [START_REF] Moskovitz | Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals[END_REF].

Although data obtained with siRNA indicate that decreased FN3K expression is toxic to cultured human fibroblasts [START_REF] Conner | Some clues as to the regulation, expression, function, and distribution of fructosamine-3-kinase and fructosamine-3-kinase-related protein[END_REF], the FN3K -/-mice that we have generated looked healthy, indicating either that the observed effects of RNAi were unrelated to depletion of FN3K or that compensatory mechanisms for FN3K deficiency exist in vivo. The conservation of FN3K in mammals and birds [START_REF] Delplanque | Tissue distribution and evolution of fructosamine 3-kinase and fructosamine 3-kinase-related protein[END_REF], indicates that it must play an important role and that animals with this enzyme have a selective advantage that can be linked either to the protein-repair activity of FN3K or to its action on free fructose--lysine. It is likely that this selective advantage, which was not apparent in animals reared in the laboratory, would only become noticeable under "stressed" conditions. Identifying the reason why protein repair is important is a difficult task, because a multitude of proteins are potential targets for protein repair mechanisms. Furthermore, the damages that are repaired occur at a number of different sites on each protein and with a low stoichiometry at each site. Conceivably, repairing lysine residues may be important to restore enzymatic activity, protein-protein interaction or recognition sites for phosphorylation (which often comprise basic residues) or ubiquitinylation. The availability of an animal model will allow for a better understanding of the physiological importance of this new repair mechanism. Whole blood extracts, erythrocyte lysates from FN3K +/+ and FN3K -/-mice (100 g per lane) or control lysozyme or glycated (25 g per lane) were separated by SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and phosphorylated with recombinant E. coli fructoselysine-6-kinase and [ - 32 P]ATP. The membrane was overloaded to enhance the signal of the faintest bands. As a result of this, the phosphorylation of the lower band, corresponding to hemoglobin monomers is underestimated compared to the other bands.

FIGURE LEGENDS

fructoselysine 6 -

 6 kinase (see Reagents) in an assay mixture (100 l) containing 25 l of extract in 25 mM Hepes, pH 7.1, 2 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 20 mM NaF, 10 M ATP-Mg and 10 000 c.p.m. [ -32 P]ATP/ l. After 10 and 15 min incubation at 30°C, the reaction was stopped by mixing samples (30 l) with ice-cold HClO 4 (200 l final volume and 5% (w/v) final concentration), neutralizing with 3M KHCO 3 and removal of the insoluble precipitate by centrifugation. The supernatant was further diluted (3fold) in 0.15 M NaCl and applied onto an AG 1-X8 column (Cl -form, 1 ml) equilibrated in 0.15 M NaCl. Fructoselysine 6-[ 32 P]phosphate was eluted in the flow-through and in the washing fractions (4 x 2 ml 0.15 M NaCl) and separated from the remaining [ -32 P]ATP. The sum of the radioactivity found in these fractions was used to calculate the concentration of fructose--lysine in the extracts.

Biochemical

  Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060684

  P] tagging of Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060684

Fig. 1 .

 1 Fig. 1. Targeted deletion in the FN3K locus in mice.In Panel A, the shaded boxes represent the exons in the FN3K gene, the grey boxes those in the FN3K-RP gene[START_REF] Collard | A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines[END_REF] and the white boxes the neomycin resistant cassette (Neo) and the -galactosidase marker ( -Gal). Key restriction sites and localization of probes used during Southern blotting are shown. Panel B confirms the correct targeting of the 5' and 3' arms and excludes any additional insertion by Southern blotting. Panel C confirms that transcription of the FN3K locus has been ablated in various tissues in mice that are homozygous for the FN3K deletion. RT-PCR analysis of the sequence spanning exons 1 (primer: 5'-CGTGTTTGTCAAGGTCAATCG-3') to 3 (primer: 5'-TGCCATCTGTTCCCCGAGC-3') shows the absence of a detectable transcript in FN3K -/-animals. Panel D, shows the FN3K activity in red blood cells, kidney and brain following the ablation of the FN3K gene in mouse. The activity of FN3K is related to the amount of protein in the extract. In red blood cells the activity was determined in extracts of 2 mice for each genotype. In kidney and brain extracts, the activity in each tissue was determined after pooling the extracts from two mice of each genotype prior to purification.

Fig. 2 .

 2 Fig. 2. Glycation of hemoglobin in wild type and FN3K -/-mice.GlcHb (total glycated hemoglobin that binds to phenylboronate affinity columns) in red blood cells from wild type, FN3K +/-and FN3K -/-male (Panel A) and female (Panel B) mice aged 1, 2, 4 and 6 month. Each data point represents the mean of four animals ± S.E.M.

Fig. 3 .

 3 Fig. 3. Phosphorylation of glycated residues from proteins in red blood cell extracts from wild type and FN3K -/-mice.

Fig. 4 .

 4 Fig. 4. HPLC profile of radioactive peptides after tryptic digestion of hemoglobin from FN3K +/+ and FN3K -/-mice phosphorylated with FN3K and [ -32 P]ATP.Hemoglobin from FN3K +/+ and FN3K -/-mice was partially purified on DEAE-Sepharose and incubated with mouse recombinant FN3K and [ -32 P]ATP for 2 h in vitro. After reduction of fructosamine 3-

Acknowledgements. We thank Kate Peel and Jean-François Cornut for their expert technical help. This work was supported by grants from the European Foundation for the Study of Diabetes, the Juvenile Diabetes Foundation, the Interuniversity Attraction Poles Programme-Belgian Science Policy and the Actions de Recherches Concertées of the French Community of Belgium. M.V.D.C. and P.J. are Chercheur Qualifié of the Belgian Fonds National de la Recherche Scientifique.

[ 32 P]phosphates with NaBH 4 , protein was precipitated in acid, resuspended in urea and digested overnight with trypsin [START_REF] Delpierre | Identification of fructosamine residues deglycated by fructosamine-3-kinase in human hemoglobin[END_REF]. Peptides were separated by reverse-phase HPLC and radioactive peaks were detected by Cerenkov counting.


Ulrike Brandt-Bohne

Douglas R Keene #

Fletcher A White

Manuel Koch
email: manuel.koch@uni-koeln.de

EGFL5: a novel transmembrane protein with a strong and developmentally regulated expression in the nervous system

Keywords: EGFL5, transmembrane protein, nervous system, glycosylation, glial cell, skin

EGFL5, a novel transmembrane protein with multiple Epidermal Growth Factor (EGF)-like repeats is predominantly expressed in the developing and adult central and peripheral nervous system.

The domain structure of EGFL5 consists of an N-terminal region with several potential O-glycosylation sites followed by five EGF-like domains, which are highly homologous to the short arms of laminins.

Following one single pass transmembrane domain, a highly conserved short intracellular domain with potential phosphorylation sites is present. The protein was recombinantly expressed and characterized as a tissue component. To further study the expression pattern, immunohistochemistry was performed and staining was detected in Purkinje cells of the cerebellum and in glial cells of the peripheral nervous system. Additional expression was observed in the epidermal layer of skin, papillae of the tongue and the epithelium of the gastrointestinal tract. By immunoelectron microscopy, EGFL5 was detected in glial cells of the sciatic nerve facing the basement membrane. EGFL5 represents a novel putative receptor, expressed in neuronal and non-neuronal tissues, that is regulated during development and could function as a guidance or signaling molecule.

INTRODUCTION

EGF-like domains are major modular components present in many proteins of the extracellular matrix (ECM). These modules are involved in cell adhesion, receptor-ligand interactions and tissue repair [START_REF] Campbell I D | Epidermal growth fctor-like modules Current[END_REF][START_REF] Engel | EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation?[END_REF][START_REF] Appella | Strucure and function of epidermal growth factor-like regions in proteins[END_REF]. The EGF molecule is a small protein of 53 amino acids with three internal disulfide bridges stabilizing its native structure. It consists of a small domain folded to an S-shaped structure and two short stretches of anti-parallel β-sheet. The localization of cysteine and glycine residues and the distance between them define its conformation [START_REF] Engel | EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation?[END_REF][START_REF] Appella | Strucure and function of epidermal growth factor-like regions in proteins[END_REF]. Homologous sequences of about 40 amino acids in one or more copies have been described in a large number of proteins [START_REF] Appella | Strucure and function of epidermal growth factor-like regions in proteins[END_REF]. A common feature is that this domain is present in the extracellular region of membrane bound or in secreted proteins, with the exception of the intracellular prostaglandin G/H synthase. In addition to these prototype EGF-like domains, laminins contain laminin type EGF-like (LE) domains, consisting of about 60 amino acids. The tertiary structure is remotely similar at its N-terminal to that of the classical EGF-like module. The two domain types differ in their number of conserved cysteines; six in classical EGF-like and eight in laminin-type EGF-like domains. The sequences between conserved cysteines vary in length [START_REF] Engel | EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation?[END_REF][START_REF] Appella | Strucure and function of epidermal growth factor-like regions in proteins[END_REF][START_REF] Martin | Basement membrane proteins: molecular structure and function Adv[END_REF].

Proteins containing EGF-like domains are thought to be involved in the development, maintenance and injury response of the nervous system. Candidates for such processes are the Notch family of proteins, reelin, slit and tenascin-C [START_REF] Lasky | Notch signaling, brain development, and human disease[END_REF][START_REF] Fatemi | Reelin glycoprotein: structure, biology and roles in health and disease[END_REF][START_REF] Fan | Slit stimulation recruits Dock and Pak to the roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline[END_REF][START_REF] Kusakabe | Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene[END_REF] Copyright 2006 Biochemical Society of neurons [START_REF] Chen | The hippocampal laminin matrix is dynamic and critical for neuronal survival[END_REF]. Mutations in these proteins have drastic effects like hypertrophy of the nervous system in the case of Notch or its ligand Delta in Drosophila or zebrafish [START_REF] De Celis | Genetic and molecular characterization of a Notch mutation in its Delta-and Serrate-binding domain in Drosophila Proc[END_REF][START_REF] Bernardos | Notch-Delta signaling is required for spatial patterning and Muller glia differentiation in the zebrafish retina[END_REF] or cerebellar and cerebral cortex disorders found in the reeler mice [START_REF] Fatemi | Reelin glycoprotein: structure, biology and roles in health and disease[END_REF]. Accordingly, human disorders like Marfan syndrome (mutations in fibrillin-1; [12]), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (Notch-3; [START_REF] Haritunians | CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand[END_REF]), Alagille syndrome (jagged 1; [START_REF] Pilia | Jagged-1 mutation analysis in Italian Alagille syndrome patients[END_REF]), and familial hypercholesterolemia (LDL receptor; [START_REF] Webb | Characterization of mutations in the low density lipoprotein (LDL)-receptor gene in patients with homozygous familial hypercholesterolemia, and frequency of these mutations in FH patients in the United Kingdom[END_REF]) arise from mutations in EGF-like domains. Furthermore, EGF-like domain containing proteins are also involved in connective tissue function and, for example, severe skin alterations (cutis laxa) can be observed if fibulin-5 is miss-expressed [START_REF] Loeys | Homozygosity for a missense mutation in fibulin-5 (FBLN5) results in a severe form of cutis laxa[END_REF]. Mutations in laminin chains (e.g. laminin α3, β3, γ2) result in skin diseases such as junctional epidermolysis bullosa [START_REF] Airenne | Structure of the human laminin gamma 2 chain gene (LAMC2): alternative splicing with different tissue distribution of two transcripts[END_REF]. The identification and characterization of further proteins containing multiple EGF-like repeats is likely to help our understanding of cell-cell interactions or ligand-receptor interactions in the nervous system and in other tissues. In this study, we describe the initial identification, characterization and expression profile of a novel transmembrane protein, called EGFL5.

EXPERIMENTAL Northern blot analysis

A 724-bp PCR product (forward 5´-dgaccccaactgccgagtcc-3´ and reverse 5´dttttaattgggtccacatggcc-3´) was labeled with 33 P-dCTP (NEN Life Science Products) using the rediprime DNA labeling system (GE Healthcare). Northern blots (CLONTECH Laboratories, Inc.) were prehybridized in 50% formamide, 5x SSPE, 1x Denhardt's, 1% SDS, 10% dextran sulfate, and 0.1 mg/ml salmon sperm DNA (Invitrogen) at 42ºC for 2 h. Without further purification, the probe was denatured in the same buffer plus 1/10 vol/vol human Cot-1 DNA (Boehringer Mannheim), and 1/10 vol/vol sheared salmon testis DNA (Invitrogen) at 94ºC for 5 min, placed on ice, added to the blots, and hybridized for 20 h. Blots were washed three times in 2x SSC, 1% SDS at 42ºC and two times in 0.1x SSC, 1% SDS at 42ºC. Blots were placed on BioMax MR film with a BioMax TranScreen-LE intensifying screen (both from Eastman Kodak Co.) for 20 h at -70ºC.

RT-PCR

RNA was isolated from animal tissues using the RNeasy kit (QIAGEN), and 1µg of total RNA was reverse-transcribed using an RT-PCR kit (CLONTECH). PCR was performed on these cDNAs using a long expand PCR kit (Boehringer Mannheim) with GADPH primers (forward: 5'dtgaaggtcggtgtgaacgga-3'; reverse: 5'-dgatggcatggactgtggtca-3') or actin primers (forward: 5´ gccattcaggcggtgctgtc; reverse: 3´ -gtgctaggtgctagggctgt) and the amount of template was normalized for each tissue. A range of cycle numbers was tested to ensure that the amounts were normalized in the linear range of the reaction. With the gene-specific primers (forward: 5'-dcccgccaccccttcggaggacgg-3'; reverse: 5'-dggccatgtggacccaattaaaactcc-3'), another PCR was performed on the normalized cDNA templates.

Expression and purification of recombinant EGFL5

The EGFL5 cDNA was generated by PCR on full length cDNA derived from mouse brain tissues using specific primers to amplify a 78 kb fragment coding for the five EGF-like domains of EGFL5. The primers used were: forward 5´-gaggctagccatatgtgtaacttctgaggttc-3´ and reverse 5´ccaggatccttacttgatgcagtttctctgaagat-3´. The sequenced construct was cloned into a modified pCEP-Pu expression vector carrying an N-terminal 6xHis tag and a thrombin cleavage site and the plasmid was transfected into EBNA 293 cells. Protein was purified from the collected cell culture supernatant using CoCl-Sepharose (CLONTECH Laboratories, Inc.) and eluted with increasing imidazole concentrations in 150 mM NaCl, 50mM Tris-HCl, pH 8. Finally, the protein was dialyzed against PBS and stored at -20°C.

Preparation of specific EGFL5 antibodies

The purified recombinant protein was used to immunize a rabbit and a guinea pig. The antisera were affinity purified on CNBr-activated Sepharose (GE Healthcare) with recombinant EGFL5 protein coupled. The specific antibodies were eluted with 150 mM NaCl, 0.1 M triethylamine, pH 11.5, and the eluate neutralized with 1 M Tris-HCl, pH 6.8 and dialyzed against PBS before freezing. The antibodies are called pAbKR18 (rabbit origin) and pAbKG20 (guinea pig origin).

Rotary shadowing and electron microscopy

For rotary shadowing experiments the protein was dialyzed against 150 mM ammonium bicarbonate. Samples were diluted to a final concentration of 70% (v/v) glycerol, sprayed onto freshly cleaved mica, and dried under vacuum (Balzers BAE 250 evaporator). Rotary shadowing was performed as described previously (Sakai et al., 2004). Replicas were examined at 80 kV in a transmission electron microscope (Philips 410LS). The protein images were measured using the imageJ (NIH, freeware) software.

Tissue preparation and extraction

Mice were sacrificed to collect tissues for analysis. Brains were dissected, weighed, extracted by homogenization in chilled buffer (1x complete, 0.2 mM EDTA, 1% NP40), and incubated on ice for 30 min. Homogenates were centrifuged at 4°C at 13000rpm for 15 min. Supernatants were mixed with 5x SDS-PAGE sample buffer supplemented with 4% β-mercaptoethanol and applied to a 10-12% SDSpolyacryamide gel.

Removal of N-linked glycans

PNGase F (Biolabs) was used to digest purified proteins and tissue extracts according to manufacturers specifications.

SDS-polyacrylamide gel electrophoresis and immunoblotting

SDS-PAGE was performed as described [18]. For immunoblots the proteins were transferred to nitrocellulose, blocked in TBS containing 0.05% Tween and 3% low fat milk powder. The blots were incubated with the respective antibodies diluted in TBS containing 0.05% Tween and 3% low fat milk powder for 2 h at room temperature or over night at 4°C. Bound antibodies were detected by luminescence using peroxidase-conjugated goat anti-rabbit IgG (Sigma), goat anti-mouse IgG (DAKO) or goat anti-guinea-pig IgG (DAKO).

Tissue preparation, frozen sections and immunohistochemistry

Nerve whole mount preparations

Nerve whole mount preparations were performed with adult tissues. Nerves were dissected, treated with clostridial collagenase (3.5 mg/ml, Sigma) and dispase II (Roche Diagnostics) for 20-30 min at 37°C, separated on collagen coated coverslips, fixed with 2% paraformaldehyde for 5-10 min at room temperature and washed before immunohistochemical staining.

RN2 schwannoma cell culture and staining

RN2 rat schwannoma cells were plated and grown on chamber slides for 2 days in 10% fetal calf serum (FCS) containing DMEM/F12 medium (Invitrogen). The primary antibody was incubated with the cells for 30 min at room temperature. Cells were washed, fixed with 2% PFA for 2-5 min and permeabilized with 0.2% Triton for 1-5 min at room temperature. Cells were blocked with NGS for subsequent secondary antibody incubation. Nuclear staining was performed using bisbenzimidine (0.001mg/ml in methanol diluted in PBS, Serva), whereas the F-actin cytoskeleton of RN2 cells was detected by FITC-conjugated phalloidin (Molecular Probes).

Immunoelectronmicroscopy

Mouse sciatic nerve was diced and immersed in primary KG20 antibody diluted in serum free DMEM medium overnight at 4°C. The tissue was washed in DMEM for 4 h and immersed in a suspension of 1-nm gold labeled secondary antibody in medium overnight at 4°C. Following an extensive wash in medium, the tissue was immersed in gold enhancement solution, rinsed, fixed in 1.5% glutaraldehyde/1.5% paraformaldehyde with 0.05 % tannic acid, osmicated, and prepared for transmission electron microscopy by a standard protocol.

RESULTS

Identification and characterization of the EGFL5 genes

By screening the EST (expressed sequence tags) database for Epidermal Growth Factor like (EGFlike) domains, we identified a murine sequence with high homology to the laminin β2 chain and cloned the gene. The encoded type I single pass transmembrane protein, named EGFL5, contains five EGF-like domains.

The EGFL5 genes map to syntenic regions in the human and mouse genome on chromosome 9

(9q32-q33.3) and 4 (CA2), respectively. At the 5´-end, the human and murine genes are flanked by the CDK5 regulatory subunit associated protein 2, the DBC1 (deleted in bladder cancer 1) gene, the TLR4 (toll-like receptor 4), and astrotactin 2 genes. The latter is already located in the adjacent human chromosomal region 9q31 and in murine chromosomal region 4CA1. There are no further genes downstream in the same syntenic region.

The human (XM_376905) and mouse (NM_172694) genomic sequences are fully annotated in the public database. The organization of the exon/intron size, length and codon phase is very similar in both species. The human and mouse EGFL5 genes consist of 6 exons and span over 113.66 kb and 106.09 kb, respectively. The corresponding cDNAs are about 6298 bp (human) and 6146 bp (murine) long (Figure 1). Exon 1 encodes the N-terminal signal peptide sequence and a region of unknown function, whereas exons 2-5 code for EGF-like repeats 1-5, and exon 6 a transmembrane region and a cytoplasmic tail. A large portion of exon 6 encodes an unusually long untranslated region of 4326 bp and at the 3' end a conserved polyadenylation site is present.

Analysis of EGFL5 domain structure

EGFL5 is highly conserved between different species in vertebrates; however no homologous gene can be located in the C. elegans or D. melanogaster genome. The murine sequence is highly homologous (68%) with its human counterpart (Figure 2) and the proteins have an identical domain structure. The protein sequence consists of 600 amino acid residues in both mouse and human, which yields a calculated mass of 63 kDa. An N-terminal signal peptide (amino acids 1-34) is followed by a stretch of 168 amino acids. This region is highly homologous to the EGFL5 sequences in rat, chimpanzee and human (Supplementary Figure 1A). alignments of all five EGF-like motifs were performed using the BlastP algorithm with the blosum62 matrix. In an end-weight pileUp run, the consensus sequence of conserved cysteine and glycine residues in all five EGF-like domains is shown (Supplementary figure 1B). In the human orthologue, the EGF-like domains share 81% sequence homology with the ones found in mouse. Pronounced sequence similarities are found to the laminin β1 and β2 chains while other members of the laminin group, such as the laminin α1, -2, -3, and -4 chains as well as laminin γ1 and γ3 chains display weaker homologies to EGFL5. The shared regions are restricted to the EGF-like domains of EGFL5, and multiple alignments are shown in supplementary figure 2A for the murine EGF like domains 1-5.

A stretch of 60 amino acids, which displays no significant homology to any other protein, might be a putative linker region and separates the EGF-like domains from the transmembrane region. The short transmembrane region comprises 21 amino acids, followed by a highly conserved cytoplasmic stretch.

This intracellular region is almost identical to the sequence of the human EGFL5 (98%) with only one arginine being replaced by a glycine. This unique sequence is highly conserved between the orthologous genes in different species (Supplementary figure 2B).

Prediction of posttranslational modifications

Putative posttranslational modifications can in part be predicted by analyzing the amino acid sequence for motifs similar to those described in other proteins. Eight putative N-glycosylation sites were

EGFL5 gene expression

To determine the length of EGFL5 mRNA we performed northern blot hybridization with mRNA derived from different embryonic mouse tissues and developmental stages (Figure 3A andB). The first expression of EGFL5 mRNA was detected at embryonic day E11. A variety of tissue (heart, brain, spleen, kidney and testis) gave a clear band of 7.0 kb, whereas weaker hybridization signals were found in lung and liver, and no signal in skeletal muscle. RT-PCR analysis confirmed gene expression in several regions of the brain and weaker expression in dorsal root ganglia, skin and keratinocytes (Figure 3C).

EGFL5 does not seem to be alternatively spliced, since only one single band was detected by northern blot analysis and since no alternative EST clones were identified. In silico analysis of different EST clones from expressed single sequence tag gene bank shows that EGFL5 is expressed in embryonic and adult tissues. Several clones originated from nervous tissues, especially from diencephalon, eye and ganglia. In non-neuronal tissues, numerous transcripts can be found in pancreas, lymph node and spleen, whereas few clones are from genital tracts, heart, bone and thymus.

Recombinant EGFL5 expression and antibody production

In order to characterize the EGFL5 protein, a 930 bp insert (EGFL5-EGF), which encodes the five EGF-like domains, was amplified using specific primers (see Material and Methods). (Figure 4A). The recombinant EGFL5-EGF was used for immunization of a rabbit and a guinea pig. The affinity purified polyclonal rabbit KR18 and guinea pig KG20 antibodies were highly specific and recognized the recombinant protein as well as the full-length protein in tissue extracts of brain as a single band (Figure 4B andC).

The mass of the purified EGFL5 ectodomain protein by SDS-PAGE (~75 kDa) was much higher than the 33 kDa predicted from the sequence, while MALDI-TOF mass spectrometry gave a peak at 41 kDa and after PNGase F digestion 32 kDa. Similarly, the full length protein detected by immunoblot in murine brain extracts showed a mass of ~180 kDa, in contrast to the calculated 63 kDa. The presence of N-linked glycans, predicted by sequence comparison, was confirmed by N-glycosidase F digestion, which resulted in a shift of ~30 kDa in both the recombinant EGFL5-EGF and in full length EGFL5 from brain tissue extracts (Figure 4B andC).

No Ca 2+ -binding EGF-like modules are predicted in EGFL5, and recombinant EGFL5-EGF as well as full length EGFL5 detected in brain tissue extracts show no difference in their electrophoretic mobility under either reduced or non-reduced SDS-PAGE (Figure 4D andE).

EGFL5 expression in the central nervous system

Immunohistochemistry using affinity purified antibodies raised against the recombinant EGFL5-EGF fragment showed EGFL5 protein expression throughout the E18.5 embryonic brain and a more restricted expression by postnatal day 1 (Figure 5A andB). Notably, higher protein levels were found in the outer layers of the cortex than in inner layers. High expression was detected in the CA1 region, the stratium radium, and the oriens layers of the hippocampus, whereas the nuclear layers in CA1 and CA2

were negative (Figure 5E). Additional signals were found in the diencephalon, superior and inferior colliculus, cerebellum, facial nucleus and dorsal medulla oblongata of embryonic and newborn brain.

Cranial sensory ganglia and nerves also express EGFL5 (Figure 5A andB). The cerebellar expression becomes more limited late in development (Figure 5A-C) and EGFL5 is subsequently expressed only by Purkinje cells of the adult cerebellum (Figure 5D). Staining was also detected in the olfactory bulb and olfactory epithelium (Figure 5F andG).

In eyes from the stage E12.5, positive signals were seen only in the lens (Figure 5H), whereas on postnatal day 1 (P1) EGFL5 was present throughout the lens and retinal ganglion cell layer (Figure 5I). In the adult eye, EGLF5 expression was confined to interneural layers of the retina, including the inner and outer plexiform layers, the outer region of the photoreceptor layer and the Bruchs membrane. No signal was detected in the nuclear layers or in the pigment epithelium (Figure 5J).

Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691

Copyright 2006 Biochemical Society

In the spinal cord, EGFL5 was present throughout the lamina of the dorsal horn and focally in the ventral commissure (Figure 5K). Little to no expression was detected in the ventral horn of the spinal cord.

EGFL5 expression in the peripheral nervous system

EGFL5 expression is prominent in both sensory ganglia (cranial and somatosensory) and associated peripheral nerves. Sections through embryonic mice showed strong staining in the trigeminal ganglia, dorsal root ganglia and thoracic sympathetic chain ganglia (Figure 5A, B and K-N). The immunopositive cells within these sensory ganglia are likely to represent myelinating and non-myelinating Schwann cells and satellite glia cells. Prominent staining was also present throughout the length of the peripheral nerves, including the trigeminal nerve (see also Figure 5A andB). In co-staining experiments, an overlap was seen with laminin present in basement membranes of Schwann cells ensheating peripheral nerves (not shown). EGFL5 was also detected in whole mount preparations of sciatic nerves and in sections depicting afferent fibers of the tongue and the whiskers (Figure 6J and7D).

To determine the nature of EGFL5-immunopositive peripheral glial cells, further immunostaining was performed on both longitudinal and transverse sections of sciatic nerves. Co-staining with a glia specific lectin showed that EGFL5 occurs in structures around dorsal root nerve cells, representing associated glia cells (Figure 6A andB). EGFL5 showed a colocalization with laminins containing the γ1 chain (Figure 6C) and was present on glia throughout peripheral nerves, as seen with antibodies against neurofilament heavy chain and against GFAP (Figure 6D-F). Accordingly, EGFL5 is present only on cells surrounding nerve fibers and not in the actual nerve fibers. In cell culture, EGFL5 is expressed on rat RN2 schwannoma (Figure 6G andH) and on rat C6 oligodendroglial cells (Figure 6I). Immunogold labeling of sciatic nerves with the KG20 antibodies localized EGFL5 to the outer membrane of myelinating Schwann cells (Figure 6K andL).

EGFL5 expression on keratinocytes of the skin and tongue

EGFL5 was also detected in tongue and skin epithelium. In agreement with the lower EGFL5 mRNA levels in skin compared to brain, the immunofluorescence signal in skin was also weaker than those in the nervous system (Figure 7A). In skin, all epidermal layers express EGFL5 and strong staining was also seen within the external root sheath of hair follicles (Figure 7B andC). In the tongue, the lingual filiform papillae, which cover the entire anterior part of the back of the tongue and consist of cone-shaped structures with a core of connective tissue, are EGFL5 positive (Figure 7D andE). EGFL5 was also detected in the esophageal and intestinal epithelium (Figure 7F andG).

Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691

Copyright 2006 Biochemical Society DISCUSSION EGFL5, a novel transmembrane protein, was identified by screening the EST database for proteins containing EGF-like domains. Related proteins, containing multiple EGF-like domains, often play crucial roles in the development and maintenance of the nervous system. Even though the EGFL5 gene has been detected earlier and both the murine and the human sequences submitted to the database, no detailed characterization was performed up to date. The human cDNA of EGFL5 was identified in a search for high-molecular-weight proteins with multiple EGF-like motifs using motif-trap screening methods [START_REF] Nakayama | Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening[END_REF] and the mouse gene was identified and shown to be located on chromosome 4 [20].

We could demonstrate that the human and mouse EGFL5 genes are clearly orthologous. They are located on syntenic areas of the genome, the exon-intron organization is similar and the amino acid sequences are highly conserved (68%). On the mRNA level a single EGFL5 transcript was detected, indicating the absence of alternative splice variants. Translation of the cDNA sequence reveals a signal peptide sequence. The subsequent region shows no homology to any other sequences found in the database. The middle part of EGFL5 in the extracellular region shows homology to EGF like domains.

Laminins also contain numerous EGF-like domains and represent heterotrimeric proteins consisting of three chains: one α, one β, and one γ chain [21]. By database analysis of this region, EGF repeats from the laminin β2 chain show the highest degree of conservation at the amino acid level, followed by the laminin β1 subunit, the α 1 and -2 chains, and the laminin γ1 chain precursor. The characteristics of the laminin-type of EGF-like domains are that they contain eight instead of six conserved cysteines.

Additional analysis of the EGFL5 EGF repeat reveals that no putative Ca-binding consensus sequences are present. Following the short transmembrane region, an unique intracellular stretch containing putative phosphorylation sites for signal transduction events are located at the C-terminus. This region is highly conserved between different species (about 98%).

Posttranslational modifications, such as glycosylations, were predicted at the amino acid level and could in part be verified experimentally by glycan digestion. Glycosylations have been described to be involved in folding and stability of proteins, but also in cell -cell and cell -matrix interactions [START_REF] Haltiwanger | Role of glycosylation in development[END_REF].

Especially high amounts of O-linked glycosylations of the mucin type, commonly found on mucins, were predicted for the N-terminal region of EGFL5. A general feature of mucins is the presence of regions rich in threonine and/or serine whose hydroxyl groups are in O-glycosydic linkage with the oligosaccharides involved [START_REF] Gendler | Epithelial mucin genes[END_REF]. As shown for other mucin-type glycosylated proteins [START_REF] Eckhardt | Porcine submaxillary mucin contains a cystine-rich, carboxyl-terminal domain in addition to a highly repetitive, glycosylated domain[END_REF], the C-terminal region of EGFL5 is also rich in serine, threonine, glycine, alanine, and proline, which together account for more than threefourths of the amino acid residues.

As large number of proteins that are not classical mucins carry mucin-type glycosylation [START_REF] Perez-Vilar | The structure and assembly of secreted mucins[END_REF] and are thought to be involved in recognition of other proteins or cells resulting in adhesion [START_REF] Melnick | An alternatively spliced Muc10 glycoprotein ligand for putative L-selectin binding during mouse embryonic submandibular gland morphogenesis Arch[END_REF], EGFL5 Copyright 2006 Biochemical Society might be involved in similar processes. High levels of EGFL5 transcripts and protein were detected in neuronal and glial cells of the central and peripheral nervous system. Classical mucins are thought to be absent in the nervous system, while other proteins with a mucin-type glycosylation, e.g. dystroglycans, are expressed in brain and nerves [START_REF] Michele | Dystrophin-glycoprotein complex: post-translational processing and dystroglycan function[END_REF] and have been implicated in myelination and synaptogenesis [START_REF] Chiu | Laminin and s-laminin are produced and released by astrocytes, Schwann cells, and schwannomas in culture[END_REF].

Deglycosylation of dystroglycans lead to an impairment of ligand binding and causes muscular dystrophies. In addition to the N-linked glycans predicted in the region of unknown function, six additional putative sites are present in the EGF-like motifs. We could verify the presence of N-linked glycans on both recombinant and native murine full length EGFL5, as deglycosylation of EGFL5 followed by SDS-PAGE/ immonoblot analysis resulted in a shift in mass for both. Further glycosylation and other posttranslational modifications can be expected since the size of the protein after PNGase F digestion is still higher than the calculated mass.

We generated specific antibodies against the EGFL5 mouse protein which specifically reacted with one band in brain extracts. By immunohistochemistry the main expression of EGFL5 is confined to nervous tissues. It cannot only be detected in neuronal cells in the central nervous system, but also in the peripheral nervous system. Neural cell bodies and Purkinje cells in the cerebellum as well as nerve associated cell types express EGFL5 at their surface. The latter could be identified as Schwann cells by immunofluorescent staining of tissue sections and schwannoma cells also express EGFL5. By immunogold labeling and electronmicroscopy EGFL5 was detected on the outer membrane region of myelinated Schwann cells ensheating sciatic nerves.

Similarly to laminins, EGFL5 was shown to be present in a variety of tissues, especially during development. Whereas in some tissue, like the olfactory bulb and the retina, laminin and EGFL5 stainings do not overlap; in others a close localization was observed. Also in the central nervous system, the cellular sources of the two proteins appear to overlap partially, including glia and neurons. Laminins are detected in astrocyte cultures but are absent from oligodendrocytes [START_REF] Chiu | Laminin and s-laminin are produced and released by astrocytes, Schwann cells, and schwannomas in culture[END_REF], playing a role in axon outgrowth.

Glia cells of the central nervous system also seem to express EGFL5, as staining of brain sections appear diffuse and can not always be assigned to specific neurons. In contrast to laminins, EGFL5 can be detected on cultured C6 oligodendroglia. Copyright 2006 Biochemical Society the basic axon scaffolds from which neuronal connections evolve. The importance of such interactive signaling events is illustrated by the action of glial transcription factors and of glial axon guidance cues such as netrin-1 [START_REF] Deiner | Altered midline axon pathways and ectopic neurons in the developing hypothalamus of netrin-1-and DCC-deficient mice[END_REF] and slit [START_REF] Fan | Slit stimulation recruits Dock and Pak to the roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline[END_REF], which together regulate the commissural crossing of pioneer axons at the neural midline.

In such events, the defining principle is the mutually reinforcement and dependent signaling that occurs in a network of developing neurons and glia [29]. EGFL5 could represent a guidance molecule, as its expression pattern is present during the critical time points in development. The commencing broad expression could represent a migration or differentiation signal, which becomes more confined to specific structures during development. In addition it could also play a role in maintenance and stabilization processes, as it still persists in adulthood. In addition, EGFL5 could be involved in myelination, as it is present in the basement membranes of myelinated Schwann cells in the peripheral nervous system.

  as well as laminins, which play a role in supporting the survival Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691

  As indicated by its name, EGFL5 contains five EGF-like domains (EGF1: amino acids 202-251, EGF2: 252-298, EGF3: 299-346, EGF4: 347-397 EGF5: 398-449). They match the EGF motif consensus sequence CxCxxxxxGxxC, where x represents any residue, G an often conserved glycine, and C indicates conserved cysteines, providing disulfide bridges. The EGF-like domains of EGFL5 are 47-52 amino acids in length, contain eight cysteines and are similar to the LE domains found in laminins. Multiple

  detected and are indicated by asterisks in figure 1 (NetNGlyc 1.0 server), with six being present in the EGF-like domains. Numerous putative glycosylation sites for O-linked glycans (NetOGlyc 3.1 server)were predicted within the N-terminal region and for the C-terminal stretch between the EGF-like domains and the transmembrane region (dotted lines in figure1), whereas no such O-GalNAc (mucin-type) glycosylations were predicted for the EGF-like domains or the intracellular region. In contrast, YinOYan 1.2 prediction results for O-(β)-GlcNAc sites, mainly found in intracellular or nuclear proteins, indicate a putative site at position T591, but the prediction level is just above threshold. This same threonine is identified by the motif scan results (CTAIL) with medium stringency, indicating a potential proline dependent serine/threonine kinase group for the cyclin dependant kinase-5 (CDK5). Searching with low stringency results in additional predicted sites possibly recognized by calmodulin dependent kinase-2 (T590 and S559). Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691 Copyright 2006 Biochemical Society The amino acid composition of murine and human EGFL5 shows 42 cysteine residues in the sequence, representing 7% of all amino acids. 40 out of these 42 cysteines are located in the EGF-like domains; none are present in the N-terminal region of unknown function and in the intracellular domain, while two occur in the signal peptide.The C-terminal region contains frequent serine (12.5%) and threonine (16.7%) residues, which may serve as putative O-glycosylation sites as in mucins. The amino acid composition of this unknown region shows that 75.6% out of the total 186 amino acids are accounted for by six amino acids, serine (12.5%), threonine (16.7%), proline (20.8%), glycine (8.3%), alanine (11.9%) and valine (5.4%). The proportions of positively charged residues (i.e. arginine and lysine) in the murine and human intracellular EGFL5 stretches are 16% and 15% respectively, but these are not clustered. Three putative phosphorylation sites are predicted in the intracellular region (S561, S562, and Y563) (NetPhos2.0, Supplementary figure2B), which are present in both mouse and human EGFL5. Other programs confirm this prediction indicating the SYHD sequence as a possible phosphorylation site for casein kinase II (CK2). In addition, one predicted phosphorylation site is located in the short stretch separating the EGF-like domains from the transmembrane region. Finally, the hydrophobicity plots of both human and mouse EGFL5 show hydrophobic stretches in the N-terminal region, which corresponds to the signal peptide and the Cterminal transmembrane region (www.bioinformatics.weizmann.ac.il).

  The construct was Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691 Copyright 2006 Biochemical Society transfected into EBNA 293 cells and the expressed protein purified. SDS-PAGE of the purified EGFL5-EGF protein showed a single band at ~75 kDa. Rotary shadowing experiments confirmed the homogeneity of the sample and showed 12.56 nm +/-1.3 nm long rod like particles with some bends

  Glial cells control the survival of associated neurons in both Drosophila and mammals, but this control is dependent on the prior neuronal triggering of glial cell fate commitment and trophic factor expression. Differentiating glial cells and neurons define the course of nervous system development by reciprocal interactions between the two cell types[29]. Therefore, the expression pattern of specific proteins at a critical time point may play a crucial role. EGFL5 expression is regulated during development, with protein being detectable from around embryonic day E12.5 up to the adult stage. In mammals, it has been shown that many proteins containing EGF-like repeats play a role in both events. Similarly, early differentiating neurons and their associated glia rely on reciprocal signaling to establish Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691

Figure 1 :

 1 Figure 1: Exon/intron organization of the human and murine EGFL5 genes and domain structures of the EGFL5 proteins. The genes are about 110 kb long and consist of six exons. Potential Nglycosylation sites are marked by asterisks, whereas predicted O-glycosylation sites are marked by dotted lines. SP, signal peptide; TM, transmembrane; CT, cytoplasmatic tail.

Figure 2 :

 2 Figure 2: Homology of the mouse and human EGFL5 amino acid sequences. The signal peptide (SP) is underlined, the unknown region is shown in dotted boxes, the EGF-like domains in closed boxes, and the transmembrane region in a dashed box. Identical amino acid residues are marked in grey. m, mouse; h, human.

Figure 3 :

 3 Figure 3: Northern blot and RT-PCR analysis. A: Northern blot analysis of murine tissues detects EGFL5 mRNA as a single band at ~7.4 kb in brain. Weaker signals at the same size are present in nonneuronal tissues as heart, spleen, liver, kidney, and testis. B: mRNA levels are regulated during development. C: Semi-quantitative RT-PCR analysis depicts different levels of mRNA in neuronal tissues and in skin.

Figure 4 :

 4 Figure 4: Structural analysis of EGFL5 protein. A: Rod like particles representing the recombinant EGFL5-EGF protein were detected by rotary shadowing electron microscopy. B: The affinity purified rabbit anti-EGFL5 antibody KR18 recognizes the recombinant protein at ~75 kDa in SDS-PAGE immunoblot. PNGase F digestion results in a mass shift of ~30 kDa. C: EGFL5 is detected as a single ~180 kDa band in brain extract that shifts to ~150 kDa upon PNGase F digestion. The electrophoretic mobility is analyzed under reduced or non-reduced condition in E: recombinant EGFL5-EGF and F: a brain tissue extract detected with the KR18 antibody by immunoblotting.

Figure 5 :

 5 Figure 5: Protein expression in the central nervous system. EFGL5 expression is detected in all structures of (A) embryo (E18.5) and (B) newborn brain (P1). The strongest staining is in (C) cerebellum (E18.5), mesencephalon, hindbrain, and in the hippocampus (E: adult). (D) In the adult cerebellum expression is restricted to Purkinje cells (D; adult). Facial nuclei, dorsal medulla oblongata, trigeminal ganglia and nerves also express EGFL5 (A and B, respectively, see arrows), as do cells of the olfactory bulb (F; adult) and olfactory epithelium (G; P1). Further signals are found in the developing eye. (H) At stage E12.5 protein expression can be detected in the anterior lens and by P1, throughout the lens and inner retinal layers (I). (J) In adult eyes, the staining is restricted to specific cell layers, e.g. as the inner and outer plexiform layer, the photoreceptor layer and Bruchs membrane. (K) EGFL5 is expressed in

Figure 6 :

 6 Figure 6: EGFL5 protein expression in the peripheral nervous system. EGFL5 (red) staining is (A) present in satellite glia cells and (B) around dorsal root nerves co-stained with IB4 (green), a marker for non-myelinated fibers. (C) EGFL5 expression overlaps with that for laminins containing the γ1 chain present in the basement membranes of Schwann cells. (D, E) Labeling of EGFL5 and neurofilament 200 kDa (green) in sciatic nerves shows that EGFL5 is expressed in myelinating Schwann cells. EGFL5 and (F) GFAP, expressed by subsets of Schwann cells, also show a limited overlap. In cell culture, (G, H) both RN2 schwannoma cells (H: costain for F-actin) and (I) C6 oligodendroglia cell lines are both positive for EGFL5. (J) EGFL5 expression in peripheral nerves is detected in whole mount preparations of trigeminal nerves. (K, L) Electron microscopy images of immunogold labeled sciatic nerve sections. EGFL5 is present close to the basement membrane of myelinating Schwann cells.

Figure 7 :

 7 Figure 7: EGFL5 protein expression in skin and other keratinized tissues: On cryosections of embryonic and newborn tissues, EGFL5 is detected in (A-C) skin and around hair follicles (P1), (D, E) in keratinocytes of the tongue (P1), (F) oesophagus (E16.5), and (G) intestine (E16.5). TriG, trigeminal ganglia; TriN, trigeminal nerve; Wh, whiskers; Hf, hair follicle; olfE, olfactory epithelium; K, keratinocytes.

Fig. 2

 2 Fig. 2:

Biochemical Journal Immediate Publication. Published on 18 Sep 2006 as manuscript BJ20060691

  cells of the spinal cord, the dorsal horn, and the ventral root (E19.5). Additional staining is detected in dorsal root ganglia (L), thoracic sympathetic chain ganglia of E19.5 embryos (M), and DRGs of adult

	animals (N). Co, Cortex; Po, posteriour thalamic nuclei group; DpMe, deep mesencephalic nuclei; Pn,
	pontine nuclei; Fn, facial nuclei; TriN, trigeminal nerve; TriG, trigeminal ganglia; hip, hippocampus;
	LPG, lateral paragigantocellular nuclei; Mo, medulla oblongata; Sp5C, spinal 5 nuclei caudal part; Cpu,
	caudate putamen striatum; DG, dentate gyrus of hippocampus; SC, superior colliculus; IC, inferior
	colliculus; C, cerebellum; PC, Purkinje cells of the cerebellum; CA1 and CA2, field CA1 and CA2 of
	hippocampus; Mi, mitral cell layer of olfactory bulb; Gro, granular cell layer of the olfactory bulb; lo,
	lateral olfactory tract; EPI, external plexiform layer of olfactory. bulb; RGCl, retinal ganglion cell layer;
	IPL; INL, inner plexiform and nuclear layer; OPL; ONL, outer plexiform and nuclear layer; PRl,
	photoreceptor layer; BM, Bruchs membrane; PE, pigment epithelium, SC, spinal cord; DHSC, dorsal
	horn of spinal cord; VHSC, ventral horn of spinal cord; DRG, dorsal root ganglion; VR, ventral root;
	SCG: sympathetic chain ganglion.

ACKNOWLEDGEMENTS

We would like to thank Wilhelm Stoffel for the kind donation of RN2 schwannoma and C6 glial cells, and Markus Plomann and Mats Paulsson for the helpful discussion. This work was supported by the Center for Molecular Medicine Cologne, the Federal Ministry of Education and Research in Germany, an award from the Alexander von Humboldt Foundation, the Federal Ministry of Education and Research in Germany, and by Köln Fortune.