Martine Pastuglia

Véronique Ruffio-Châble

Valérie Delorme

Thierry Gaude

Christian Dumas

J Mark Cock

A Functional S Locus Anther Gene is Not Required for the Self-Incompatibility Response in Brassica oleracea

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

During compatible pollination in Brassica, pollen grains adhere to the papillar cells of the stigma, hydrate, and then germinate to emit a pollen tube. The pollen tube grows down through the transmitting tissue carrying the male gametes, which fertilize an ovum in an ovary at the base of the pistil. In self-incompatible plants, self-pollen grains are recognized by the papillar cells, and the pollination process is rapidly blocked, usually before the hydration or germination stage (reviewed in [START_REF] Nasrallah | Signalling the arrest of pollen tube development in self-incompatible plants[END_REF][START_REF] Mccubbin | Molecular mechanisms of self-incompatibility[END_REF].

Self-incompatibility (SI) in Brassica is controlled by a single, highly polymorphic locus, the S locus, of which there are more than 50 alleles [START_REF] Ockendon | Distribution of self-incompatibility alleles and breeding structure of open-pollinated cultivars of Brussel sprouts[END_REF][START_REF] Ockendon | An S-allele survey of cabbage (Brassica oleracea var capitata)[END_REF]. Rejection of self-pollen occurs when the same S allele is expressed in both pollen and stigma. The effectiveness of the SI response varies depending on which S alleles are present and the dominance/recessivity relationships that exist between different S alleles. Based on these phenomena, S alleles have been broadly classified into two groups depending on whether they tend to be dominant or codominant and confer a strong SI response (class I) or whether they tend to be recessive and confer a weak SI response (class II).

The S locus has a complex molecular structure. A number of genes have been shown to be genetically linked to the S locus: SLG (for S locus glycoprotein; [START_REF] Nasrallah | A cDNA clone encoding an S-locus specific glycoprotein from Brassica oleracea[END_REF][START_REF] Nasrallah | Amino-acid sequence of glycoproteins encoded by three alleles of the S-locus of Brassica oleracea[END_REF], SRK (for S locus receptor kinase; [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], SLA (for S locus anther; [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF], 298 and 299 [START_REF] Boyes | The selfincompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin[END_REF], and SLL 1 and SLL 2 (for S locus-linked genes; [START_REF] Yu | Molecular characterization of the S locus in two self-incompatible Brassica napus lines[END_REF]. Moreover, pulse field gel electrophoresis analysis suggests that the polymorphic nature of the S locus may extend to a region of up to 570 kb surrounding the S locus genes [START_REF] Boyes | Physical Linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea[END_REF]. As a result of this complex structure and the fact that several of the S locus genes have been implicated in the SI response, the term "S haplotype" is used rather than "S allele" to describe different allelic forms of the S locus [START_REF] Boyes | Physical Linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea[END_REF].

Two S locus genes, SLG and the SRK, are thought to be involved in perception of selfpollen by the stigma. Both genes are highly polymorphic, and both are expressed specifically at the surface of mature stigmas. Low levels of mRNA for these two genes are also present in anthers [START_REF] Sato | Activity of an S locus gene promoter in pistils and anthers of transgenic Brassica[END_REF][START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], but the protein products of the genes have not been detected in this tissue (Delorme et al., 1995b;[START_REF] Stein | SRK, the stigmaspecific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. Sequence data are available for several different alleles of both SLG and SRK. These genes exhibit the high level of polymorphism expected for genes involved in the SI response and, moreover, the different alleles of SLG and SRK can be divided into two classes based on sequence similarities, which correspond closely with the two classes defined on the basis of phenotype. Further evidence that SRK is involved in the SI response has come from the identification of self-compatible Brassica plants that also possess mutated SRK genes [START_REF] Goring | An S receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Nasrallah et al., 1994a).

SLG encodes an abundant, secreted glycoprotein [START_REF] Nasrallah | A cDNA clone encoding an S-locus specific glycoprotein from Brassica oleracea[END_REF][START_REF] Nasrallah | Amino-acid sequence of glycoproteins encoded by three alleles of the S-locus of Brassica oleracea[END_REF], whereas SRK encodes a membrane-spanning protein kinase with a predicted extracellular domain that shares extensive sequence identity (around 90%) with SLG [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF]Delorme et al., 1995b). SRK is a member of the large receptor-like kinase (RLK) gene family that has been identified in plants [START_REF] Becraft | CRINKLY4: a TNFR-like receptor kinase involved in maize epidermal differentiation[END_REF][START_REF] Braun | Plant transmembrane receptors: new pieces in the signalling puzzle[END_REF][START_REF] Hervé | Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain[END_REF]Schulze-Muth et al., 1996;[START_REF] Swarup | A new class of receptor-like protein kinase gene from Arabidopsis thaliana possessing a domain with similarity to plant lectin genes[END_REF][START_REF] Wang | The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins[END_REF]. The different gene products encoded by members of the RLK superfamily are thought to function as receptors based on their similarity to animal receptor kinases, but this has yet to be demonstrated experimentally. The SRK gene product is perhaps the best characterized member of this family. Monoclonal antibodies raised against a peptide based on the SRK sequence detected a 120-kD protein in stigma extracts, and this protein has been shown to be glycosylated and anchored in the plasma membrane (Delorme et al., 1995b;[START_REF] Stein | SRK, the stigmaspecific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. The kinase domain of SRK has been expressed in Escherichia coli and has been shown to have protein serine/threonine kinase activity [START_REF] Goring | The S-locus receptor kinase gene in a selfincompatible Brassica napus line encodes a functional serine/threonine kinase[END_REF]. A soluble, truncated form of SRK (eSRK for extracellular form of SRK) has also been detected in stigma extracts [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF]. This protein, which is produced as a result of alternative splicing of SRK transcripts, lacks the membrane-spanning and kinase domains of SRK and therefore closely resembles SLG.

Current models describing the pollen-pistil recognition step of the SI response propose that SRK, present in the cell membrane of papillar cells on the stigma surface, binds to a pollen-borne ligand and initiates a protein phosphorylation cascade that leads to the SI response. SLG and eSRK are thought to modulate signal transduction via SRK. Identification of a ligand bound by SRK would greatly facilitate testing of these models. To date, none of the proteins encoded by members of the RLK superfamily have been shown to bind a ligand (hence the term receptor-like kinase) although, in the case of SRK, two candidate ligands have been proposed in the literature.

The first candidate is a small protein (PCP7 for 7-kD pollen coat protein) present on the surface of Brassica pollen (in the pollen coat). It has been shown to interact with SLG in vitro [START_REF] Doughty | Interaction between a coating-borne peptide of the Brassica pollen grain and stigmatic-S (selfincompatibility)-locus-specific glycoproteins[END_REF]. It is thought that PCP7 or a related protein might therefore bind to SRK, either directly as a result of SRK's similarity with SLG, or indirectly as a complex incorporating SLG. However, no PCP has yet been shown to bind specifically to the gene products of a particular S haplotype and, although a cDNA clone has been isolated for one member of the PCP gene family, no members of this gene family have yet been shown to be linked to the S locus [START_REF] Stanchev | Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions[END_REF].

A second candidate for the male component of the SI recognition system is the product of the SLA gene, which was originally identified in the S 2 haplotype where it is located just downstream of the SLG 2 gene [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF]. SLA encodes two complementary transcripts that accumulate specifically in anthers. One of the transcripts contains two small open reading frames (ORFs), and it has been proposed that a peptide encoded by SLA might interact, either directly or indirectly, with SLG and SRK to initiate the SI response [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF]. A second SLA allele was shown to be interrupted by a large insertion resembling a retrotransposon in a self-compatible Brassica napus line suggesting a correlation between the presence of a functional SLA gene and SI [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF]. SLA was not detected in a number of other S haplotypes, and it has been suggested that this might be due to a high level of polymorphism between SLA alleles [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF], as would be expected for a gene involved in the SI response.

In this study, we have characterized the SLA gene from the self-compatible B.

oleracea line P57Sc and have shown that it also contains a large insertion resembling a retrotransposon. However, the structure of the SLA gene in a number of B. oleracea var botrytis lines, which carry the same S haplotype as P57Sc but which exhibit different levels self-incompatibility, indicated that a functional SLA gene is not required for the SI response.

RESULTS

Cloning of SLG and SLA from a self-compatible B. oleracea line

The cloning and characterization of a cDNA clone, denoted CG15, from the B. oleracea line P57Sc has been described previously [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. Based on sequence similarity to previously characterized SLG genes, CG15 was thought to correspond to the SLG gene of the P57Sc line (SLG Sc ; [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF].

In this study, a 880-bp BamHI fragment from the 5' end of the CG15 cDNA was used to probe 10 6 plaque-forming units of a P57Sc genomic library. Subcloning and DNA sequence analysis of phages, which hybridized with the probe, revealed that they included one clone with 100% sequence identity to CG15 and a second clone, denoted CG514, that was highly similar but not identical to CG15. A 10.5-kb region of the latter clone was sequenced and shown to share extensive similarity with the SLG/SLA region of the S 2 haplotype (Figure 1). Based on this similarity, the two genes carried by the CG514 clone have been designated SLG Sc and SLA Sc , and hence, the gene represented by CG15 is no longer denoted SLG Sc, as was originally proposed [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. We now propose that CG15 corresponds to a second, S locus-linked gene and may represent an alternative transcript of the SRK Sc gene encoding a truncated form of SRK analogous to the eSRK protein identified in the S 3 haplotype [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF].

Comparison of the SLG and SLA genes of the S Sc and S 2 haplotypes

Alignment of the SLG/SLA gene region of the S locus of the P57Sc line (S Sc ) with the corresponding region of the S 2 haplotype showed that the two sequences are highly similar, apart from an insertion of 4826 bp in SLA Sc that is described in more detail below. SLG 2 has been shown to encode two alternative transcripts, which encode the soluble SLG 2 protein and a membrane-anchored form of SLG 2 , mSLG 2 , respectively. The SLG Sc gene possesses both of the exons present in the SLG 2 gene and, therefore, potentially encodes both SLG and mSLG proteins. The deduced amino acid sequences of SLG Sc and SLG 2 share 98.2% similarity. In both the S Sc and S 2 haplotypes, SLA is located directly downstream of SLG (Figure 1). [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] reported the sequences of cDNAs corresponding to two overlapping transcripts of the SLA 2 gene: a spliced transcript that was transcribed from the same strand as SLG 2 and that contains two ORFs and an unspliced transcript that was transcribed from the opposite strand. Sequences corresponding to both of these transcripts are present in SLA Sc and exhibit 99.6 % nucleotide similarity with the spliced and unspliced transcripts of SLA 2 (Figure 1). The two ORFs in the spliced transcript are identical between the two alleles, except that the eighth codon of ORF2 is an alanine codon (GCG) in SLA 2 and an arginine codon (CGC) in SLA Sc .

The 4826-bp insertion in SLA Sc contains all the structural features of a retrotransposon (Figure 2; reviewed by [START_REF] Grandbastien | Retroelements in higher plants[END_REF]. The insertion contains two long terminal repeats (LTRs) that differ by 7-bp at a single position 32 bp from the 3' end. The LTRs are flanked by a duplicated 6-bp sequence of the SLA gene resembling the direct repeats found at sites of transposon insertion. Between the two LTRs is a region of 4432 bp that is flanked by sequences resembling sites involved in priming reverse transcription of known retrotransposons (Figure 2A). These sites consist of an 11-bp region identical to the initiator methionine tRNA from Arabidopsis at the 5' end of the internal domain and a purine-rich sequence at the 3' end.

The 4432-bp internal domain contains two long ORFs (Figure 2B). ORF1 is predicted to encode an 1132-amino acid polypeptide that includes regions similar to conserved motifs found in retransposons (Figure 2B): an RNA binding site involved in positioning the tRNA during the initiation of reverse-transcription; a protease domain implicated in the endoproteolytic cleavage of the polypeptide; an integrase region necessary for the integration of the retroelement in the host genome, and a reverse transcriptase domain. ORF2 is located 64 bp downstream of the end of ORF1 and is predicted to encode a 253 amino acid protein with an RNaseH domain. Comparison of the sequence of this element, which we have named

Melmoth [START_REF] Maturin | Archibald Constable and Company)[END_REF], with previously identified retrotransposons showed that it is most closely related to the Ty1/copia family of retroelements (Figure 2B).

A DNA fragment from within the Melmoth element (see Figure 1) was used to probe a DNA blot of restriction endonuclease digested genomic DNA of the B. oleracea var acephala and B. oleracea var botrytis (cauliflower) lines used in this study. The results indicated the presence of between one and three copies of the Melmoth sequence per haploid genome, depending on the genotype (data not shown). These results suggest that the Melmoth element is not replicating at a high frequency in the genome of the lines analyzed and it is possible that the element is an inactive member of the Ty1/copia family. However, low copy number does not necessarily indicate that an element is inactive; the maize retrotransposon Bs1,

which is present at only two to three copies in the genome, is nonetheless active because it was isolated following a transposition event [START_REF] Jin | Structure and coding properties of Bs1, a maize retrovirus-like transposon[END_REF]. On the other hand, the 7-bp difference between the two LTRs of Melmoth indicate that its insertion into SLA was not recent. The mechanism of transposition of yeast and insect retrotransposons has been well characterized and involves the formation of a terminally redundant, circular RNA molecule and, hence, the production of retrotransposons with identical LTRs. Assuming that Melmoth is mobilized by a similar mechanism, the difference between the two LTRs is likely to have been acquired subsequent to its insertion into the SLA gene. It remains to be determined whether this difference prevents subsequent transpositions of this element.

SLG Sc and SLA Sc are linked to the S locus

To demonstrate that SLG Sc and SLA Sc are linked to the S locus, we analyzed an F 2 population of 23 plants resulting from a cross between the homozygous P57Sc line and a line homozygous for the S 3a haplotype. The segregation of the two S haplotypes was followed by immunoblot analysis of stigma proteins separated by isoelectric focusing. A battery of antibodies raised against both class I and class II SLG proteins which had been developed in our Lyon laboratory for the purpose of determining the S haplotype of Brassica plants (Delorme et al., 1995a), was used for this analysis. Segregation of SLA Sc was followed by polymerase chain reaction (PCR) amplification of SLA Sc genomic DNA sequences using oligonucleotides OSLA1 and OSLA2 (see Figure 1 for the positions of the oligonucleotides relative to the SLA sequence). Figure 3 shows that an SLA Sc PCR product was only amplified from genomic DNA of F 2 progeny carrying the S Sc haplotype, indicating that SLA Sc is linked to the S locus in the P57Sc line.

Analysis of self-compatible and self-incompatible B. oleracea var botrytis lines carrying transposon-mutated SLA genes

To investigate further the role of SLA in SI, we analyzed a number of B. oleracea var botrytis lines also carrying class II S haplotypes. The origins and SI phenotypes of the B. oleracea var botrytis lines are described in Table 1. Surprisingly, despite the fact that the different lines exhibited different levels of self-incompatibility, several lines of evidence indicate that all of the B. oleracea var botrytis lines carry the S Sc haplotype. Monoclonal antibody (MAb) 157-35-50, an antibody raised against a peptide corresponding to the N terminus of the SLG protein of the P57Sc line [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF], recognizes SLG proteins from a range of class II S haplotypes, and closely related alleles can be distinguished based on the isoelectric points of the immunodetected proteins when separated by isoelectic focusing (Delorme et al., 1995a). Because the different B. oleracea var botrytis lines exhibited different levels of SI, we were interested in determining whether their SLA genes would be interrupted by an insertion.

Therefore, PCR amplification was carried out using oligonucleotides OSLA1, OSLA2, and OSLA3 (see Figure 1 for oligonucleotides). This combination of oligonucleotides was predicted to give a 798-bp PCR product in the presence of an SLA gene carrying the putative retrotransposon and a 612-bp PCR product if an uninterrupted SLA gene was provided as template. PCR amplification from genomic DNA of each of the B. oleracea var botrytis lines resulted in a product of 798-bp indicating that all seven carried an SLA gene interrupted by the putative retrotransposon (Figure 4B). The fact that the size of the PCR products amplified from DNA of the seven B. oleracea var botrytis lines was the same as that amplified from DNA of the P57Sc line (Figure 4B) indicated that the putative retrotransposon was inserted at the same position in the SLA genes of all these lines. The smaller, 612-bp amplification product obtained with S 2 DNA is consistent with the absence of an insert in the SLA 2 gene. No amplification product was obtained from DNA of the S 5 haplotype. This is consistent with the observation of [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] that this haplotype did not contain sequences which hybridised to an SLA 2 probe .

The experiment shown in Figure 4B demonstrated that all of the B. oleracea var botrytis lines, including the strongly self-incompatible lines 11035, 24062, and 41865, carried an interrupted SLA gene. It was therefore important to determine whether the insertion interfered with expression of SLA. In the S 2 haplotype, which contains a wild-type SLA gene lacking an inserted retrotransposon sequence, [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] have shown that the 1.56-kb, spliced SLA transcript accumulates in young anthers, with a maximum abundance in 7-to 9-mm long flower buds (which correspond to the bicellular and tricellular stages of microspore development) and that the 2.17-kb antisense unspliced transcript accumulates later and is most abundant when the flower buds are more than 9-mm long (anthers containing tricellular microspores and mature pollen). The increase in abundance of the unspliced transcript is concurrent with a decrease in abundance of the spliced transcript.

The site of insertion of the putative retrotransposon in SLA Sc corresponds to a region that is transcribed in both directions in SLA 2 . The corresponding site in SLA 2 is 198 bp from the 3' end of the unspliced transcript and 492 bp from the 5' end of the spliced transcript. An insertion in this region would be expected to perturb synthesis of both of the transcripts, particularly of the spliced transcript that is thought to encode a peptide product [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF].

We used RNA gel blot analysis to assay for the presence of SLA transcripts in different Brassica lines (Figure 5). Poly(A) + RNA was extracted from anthers at developmental stages corresponding to the stages when either the spliced or the unspliced transcripts were most abundant in the S 2 haplotype studied by [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF].

An RNA gel blot was probed with a DNA fragment from a region of the SLA Sc gene corresponding to a region that is transcribed in both directions in

SLA is a single copy, homozygous gene in the Brassica lines used in this study

Other possible explanations for the self-incompatible phenotype of some of the B. oleracea var botrytis lines are either that they were heterozygous at the SLA locus or that SLA is a member of a functionally redundant gene family, with only one active member being required for the SI response. All seven of the B. oleracea var botrytis plants were derived from lineages that had undergone several generations of self-fertilization (Table 1); therefore they were highly likely to be homozygous at the SLA locus. To confirm this, the seven B. oleracea var botrytis plants were self-fertilized and the segregation of SLA was followed in the progeny. Genomic DNA was prepared from the progeny, and the SLA gene structure was analyzed by PCR amplification using oligonucleotides OSLA1, OSLA2, and OSLA3. All of the progeny analyzed (a total of 117 representing between 10 and 29 progeny per line depending on the line) carried the SLA allele interrupted by an insertion (Table 2). These data provide strong evidence that the parental plants were homozygous at the SLA locus, for example a probability of homozygosity of greater than 99% was calculated for the three strongly self-incompatible B. o. var botrytis lines 11035, 24062, and 41865 (assuming a normal Mendelian segregation of SLA alleles).

Moreover, there is no evidence that SLA is part of a gene family. [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] showed that SLA is a single-copy gene in the genome of a line homozygous for the S 2 haplotype. We have confirmed this result and have shown that SLA is also single-copy gene in P57Sc and the seven B. oleracea var botrytis lines. Figure 6 shows that a DNA probe derived from the SLA gene (see Figure 1 oleracea var botrytis lines, again supporting the hypothesis that these lines carry the same S haplotype.

DISCUSSION

In this study, we have characterized an allele of the S locus-linked gene SLA from the S Sc haplotype carried by the B. oleracea var acephala line P57Sc. This allele, SLA Sc , was shown to be highly similar to the SLA 2 allele from the S 2 haplotype [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF], except that it contained a large insertion of 4826-bp that resembled retrotransposons of the Ty1/copia family (Figures 1 and2). Analysis of the structure of SLA Sc provided conflicting evidence concerning the role of SLA in the self-incompatible response. Plants homozygous for the S Sc locus have been shown to exhibit a self-compatible phenotype [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF].

The presence of an insertion in the SLA gene of this haplotype therefore provided a correlation between the self-compatible phenotype and the presence of a mutated SLA gene.

However, the high degree of similarity between SLA Sc and SLA 2 is not consistent with the proposed highly polymorphic nature of SLA [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF]. Genes involved in the recognition step of the SI response are expected to be highly polymorphic, with a different allele existing for each S haplotype. [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] proposed that SLA was not detected in haplotypes other than S 2 (and the closely related B. napus haplotype S 2W ) because a high level of polymorphism prevented cross-hybridization of probes. However, comparison of the S 2 and S Sc haplotypes showed that the nearby SLG genes had diverged to a greater extent (98.6% nucleotide similarity) than the two SLA genes (99.6% nucleotide similarity)

with SLA exhibiting a very low level of polymorphism (Figure 1).

To further investigate the role of SLA in the SI response, we analyzed a number of B.

oleracea var botrytis lines carrying S Sc or a very closely related S haplotype but exhibiting different degrees of self-fertility (Figures 3 and4; Table 1). All of these lines analyzed carried a single-copy homozygous SLA gene that was interrupted by an insertion (Figure 6). No SLA transcripts were detected in anthers of B. oleracea var botrytis line 11035, which nonetheless exhibited a strong self-incompatible response. These data strongly indicate that a functional SLA gene is not required for the SI response in B. oleracea.

The S locus is predicted to encode both the male and female components of the SI recognition system, and several studies have been initiated recently to identify the gene encoding the pollen component by analysis of the regions flanking the SLG and SRK genes of different S haplotypes. [START_REF] Boyes | The selfincompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin[END_REF] analyzed the regions downstream of the SLG 8 and SLG 13 alleles of B. campestris and B. oleracea, respectively, for sequences that exhibited haplotype specificity and anther-specific expression in a search for functional homologs of SLA 2 . No such sequences were found. This supports our hypothesis, based on the high degree of similarity between SLA 2 and SLA Sc , that SLA exhibits a low level of polymorphism and indicates that SLA is not present at the S locus in all S haplotypes.

In the same study, [START_REF] Boyes | The selfincompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin[END_REF] identified two novel genes, 298 and 299, downstream of SLG 8 in B. campestris. These genes were not characterized in detail, but the fact that both genes are expressed in leaves as well as floral organs and that gene-specific probes hybridize equally strongly with DNA from lines carrying the S 8 , S 2 or S 13 haplotypes argues against these genes being involved in determining the specificity of the SI response.

Similarly, [START_REF] Yu | Molecular characterization of the S locus in two self-incompatible Brassica napus lines[END_REF] Interestingly, a retrotransposon-like sequence has also been identified at the S locus in Nicotiana alata (Royo et al., 1996). N. alata possesses a gametophytic SI system in which pollen rejection is mediated by stigmatic S RNases. There is no known sequence homology between the S locus genes of gametophytic and sporophytic SI systems. However, in both cases, sequence polymorphism between alleles is known to extend for several kilobases into the regions surrounding the S locus genes, indicating that there has been extensive

rearrangements in these regions of the respective genomes [START_REF] Clark | Sequence variability and developmental expression of S-alleles in self-incompatible and pseudo-self-compatible petunia[END_REF][START_REF] Coleman | The flanking regions of two Petunia inflata S alleles are heterogeneous and contain repetitive sequences[END_REF][START_REF] Yu | Molecular characterization of the S locus in two self-incompatible Brassica napus lines[END_REF][START_REF] Boyes | The selfincompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin[END_REF]. It will be interesting to determine whether the presence of putative retrotransposons at two different S loci is coincidental or whether their presence is related in some way to the unusual structure of S loci. [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] have described an SLA allele (SLA 2w ) from B. napus var Westar that, like SLA Sc , contains a large insertion within the transcribed part of the gene.

Although no sequence data was provided, the description of this allele indicates that it strongly resembles the SLA Sc allele described above. The B. napus allele is greater than 99% identical to SLA 2 , apart from a large insert of 4827 bp (SLA Sc contains an insert of 4826 bp).

In particular, the two ORFs are highly conserved between SLA 2w and SLA Sc , with only one amino acid difference between each ORF. No SLA transcripts were detected in B. napus plants carrying SLA 2w by RNA blot analysis [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF].

Evidence has been available for some time indicating that the S Sc haplotype is present in the genome of B. napus var Westar. [START_REF] Robert | Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus[END_REF] have described a full-length cDNA from this variety which has a nucleotide sequence 100% identical to that of the CG15 cDNA isolated from the B. oleracea P57Sc line [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. [START_REF] Robert | Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus[END_REF] named the cDNA SLG WS2 ; however, based on our analysis of the P57Sc line, this cDNA, like CG15, most likely corresponds to an alternative transcript of SRK [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF] unpublished data). Based on these data, it seems likely that the mutated SLA gene identified by [START_REF] Boyes | An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts[END_REF] is part of an S haplotype closely related to the S Sc haplotype in B. napus and, for the reasons described above, it would, therefore, seem unlikely that the selfcompatible nature of Westar is related to the presence of a nonfunctional SLA gene. However, the fact that an S haplotype closely related to the S Sc is widespread in self-compatible B.

napus is interesting considering that this haplotype is associated with a self-compatible phenotype in some lines of B. oleracea [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF] this study). In addition, the S Sc haplotype carried by the B. oleracea P57Sc line is particularly interesting in that genetic crosses indicate that the male component of the SI response is defective. Further work will be aimed at determining the molecular basis of the self-compatible phenotype associated with this haplotype.

METHODS

Plant Material, Genetic Crosses, and Determination of Incompatibility Phenotype

The self-compatible B. oleracea var acephala line P57Sc has been described previously [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]). An F 2 population obtained by self-fertilization of an S 3a /S Sc F 1 hybrid (the parental lines are described in Delorme et al., 1995a, andGaude et al., 1993, respectively) was used to demonstrate linkage of SLA with the S locus. The incompatibility phenotypes of the F 2 progeny were determined by self-pollination and by crosses to tester plants using previously described procedures (Delorme et al., 1995b). Of the seven B.

Genomic DNA Cloning and DNA Sequencing

The SLG Sc /SLA Sc gene region was isolated from a genomic library constructed with DNA of the P57Sc line. The construction and screening of the library were as described previously (Delorme et al., 1995b). Restriction endonuclease digested fragments of isolated λ clones were subcloned into pBluescript II SK + (Stratagene, La Jolla, CA) and sequenced using the dideoxynucleotide chain termination method [START_REF] Sanger | DNA sequencing with chain termination inhibitors[END_REF] either on an automatic sequencer (Applied Biosystems, Foster City, CA) or using a T7 DNA polymerase sequencing kit (Pharmacia Biotech, Uppsala, Sweden). Sequence data was analysed using Lasergene sequence analysis software (DNASTAR, London, UK). The results of comparisons are expressed as percentage similarity, which is calculated as follows: 100 times the number of matched amino acids, divided by the sum of the length in amino acids of the aligned region plus the number of gaps introduced to optimise the alignment.

Protein Extraction, Electrophoretic Analysis, and Immunodetection of Proteins

Protein extraction, separation of proteins by isoelectric focusing or SDS-PAGE, electrotransfer onto nitrocellulose membranes and detection of antigen with antibodies were as described previously [START_REF] Gaude | Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea[END_REF][START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF]. Monoclonal antibody (MAb) 157-35-50 has been described previously [START_REF] Giranton | The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea[END_REF].

DNA Extraction, Polymerase Chain Reaction Amplification and DNA Gel Blots

Genomic DNA for DNA gel blot analysis and polymerase chain reaction (PCR) experiments was extracted as follows. Two centimeter square pieces of leaf were ground to a powder in liquid nitrogen and incubated at 65°C in 500 µL of extraction buffer (extraction buffer is 0.1 M Tris, pH 8, 50 mM EDTA, 100 mM NaCl, 1% SDS, and 9 µM 2-mercaptoethanol). After the addition of 165 µL of 3 M potassium acetate and 500 µL of a phenol-chloroform mixture

(1:1), the samples were agitated for 5 min, incubated for 10 min on ice, and centrifuged for 15 min at 18000g at 4°C. The aqueous fraction was mixed with 750 µL of isopropanol and incubated for 2 hr at room temperature. Genomic DNA was recuperated by centrifugation and washed with 70% ethanol before being resuspended in 100 µL of water.

When the DNA was prepared for PCR analysis, samples of tissue from a Brassica plant, which did not contain the template sequence for the PCR (leaves of a B. oleracea var acephala plant homozygous for the S 3 haplotype) were harvested after every fifth test sample.

These intercalated control samples underwent the same extraction and PCR amplification steps as the test samples to check that there was no cross-contamination between samples.

The oligonucleotides used for PCR amplification were: OSLA1

(5'-GAAGCCGACCCGTTTGGA-3'), OSLA2 (5'-GAGAGAAGGCGGAGGTGA-3'), and OSLA3 (5'-GTCCTGGCGTTTGAGAGC-3'), which correspond to SLA Sc sequences; and

SG2

(5'-GGCCTGCAGCAGCATTCAATCTGAC-3') and SG22

(5'-TGGAACCCTCAAAATCT-3'), which correspond to CG15 [START_REF] Gaude | Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea[END_REF].

For DNA blot analysis, 10 µg of genomic DNA was digested with the HindIII, fractionated on 0.8% agarose gels, and transferred to nylon membranes (Hybond N + ; Amersham) under alkaline conditions. A 1260-bp EcoRV-BamHI restriction fragment of SLA and a 860-bp EcoRV restriction fragment of the insertion in SLA were used as probes for SLA and the Melmoth element respectively. The probes are positioned with respect to the SLG/SLA region of the S Sc haplotype in Figure 1. Radiolabeled DNA probes were prepared using a random priming DNA labelling kit (Boehringer Mannheim). Filters were prehybridized and hybridized at 42°C in 50% formamide, 6xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.5% SDS, 0.1% ficoll, 0.1% PVP, 0.1% BSA, and herring sperm DNA (100 µg mL - 1 ). Identical results were obtained after washing at either low (30 min in 6xSSC, 0.1% SDS at 50°C) or high stringency (30 min in 0.1xSSC, 0.1% SDS at 50°C).

RNA Gel Blot Analysis

Developmental stages of anthers were determined by fluorescence microscopy observation of 4',6-diamidino-2-phenylindole-stained microspores and approximately correlated with bud length. Immature anthers containing unicellular and bicellular microspores were dissected from either 5 to 9 mm flower buds for B. oleracea var acephala plants or from 3 to 6 mm flower buds for B. oleracea var botrytis plants, which have proportionally smaller flowers.

Similarly, anthers containing tricellular microspores were dissected from 9 mm or 6 mm flower buds of B. oleracea var acephala or B. oleracea var botrytis plants, respectively. Total RNA extraction was as described by [START_REF] Cock | Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea[END_REF]. Poly(A) + RNA was purified by hybridization with magnetic beads coated with oligo(dT) 25 (Novagen, Inc., Madison, WI), and 16 µg of poly(A) + RNA was separated in each lane of a formaldehyde gel before being transferred to a nylon filter for hybridization. Preparation of radiolabelled probes and hybridisation conditions were as described by [START_REF] Pastuglia | Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea[END_REF]. The EMBL accession number for the SLG Sc /SLA Sc sequence is Y12321. 1985), Tst1 is from potato [START_REF] Camirand | The complete nucleotide sequence of the Tst1 retrotransposon of potato[END_REF] and Ta1-3 is from Arabidopsis [START_REF] Voytas | A copia-like transposable element family in Arabidopsis thaliana[END_REF]. DNA was extracted from two parental plants, P57Sc (S Sc ) and a plant homozygous for the S 3a haplotype (S 3a ), and from 23 F 2 progeny (F 2 ) descended from the two parental plants. PCR amplification was carried out using oligonucleotides OSLA1 and OSLA2 (see Figure 1), and the PCR products were separated on an agarose gel and stained with ethidium bromide. The number at left indicates the length of the PCR products in base pairs. Segregation of the two S haplotypes in the F 2 population was followed by immunoblot analysis of stigmatic SLG proteins separated by isoelectric focusing (Delorme et al., 1995a), and the S haplotypes carried by the different progeny are indicated below the lanes. , 24062, 11035, 17144, 40443, 14518, and 22495. A 1260-bp EcoRV-BamHI fragment from SLA Sc (SLA probe in Figure 1) was used as a probe.

FIGURE LEGENDS

Numbers at left indicate the lengths of hybridizing fragments in kilobases. a Pollen tube mean scores were determined by aniline blue staining of pistils 24 hours after pollination. Three pistils were scored at one time, and between two to five triplets of pistils were scored for each plant. Nine scores were defined: 0, 0 tubes; 1, 1 or 2 tubes; 2, 3-5 tubes;

TABLES

3, 6-9 tubes; 4, 10-14 tubes; 5, 15-25 tubes; 6, 26-50 tubes; 7, 51-100 tubes; 8 = >100 tubes.

The mean score was determined from all the pistils tested for a particular plant, min. and max. scores refer to minimum and maximum scores, respectively, obtained from a triplet of pistils for each plant.

b The SI phenotype based on pollen tube mean scores: SI, self-incompatible; SC, selfcompatible. a Genomic DNA was extracted from each of the plants tested and a fragment of SLA was amplified using oligonucleotides OSLA1, OSLA2, and OSLA3 (see Figure 1).

b The SI phenotype based on the pollen tube mean scores (see Table 1): SI, self-incompatible; SC, self-compatible.

c A 798-bp product was amplified from genomic DNA of these plants indicating the presence of an SLA gene interrupted by an insertion.

Figure

  Figure4Ashows that the pattern of stigma glycoproteins detected by

  SLA 2 . Bands corresponding to both the spliced and unspliced transcripts were detected in anther poly(A) + RNA from an S 2 homozygous line but neither SLA transcript was detected in anthers from either P57Sc or the self-incompatible B. oleracea var botrytis line 11035. The two SLA transcripts were, therefore, not detected in lines carrying an SLA Sc allele with an inserted Melmoth element, indicating that the presence of this element prevents expression of the SLA gene. The absence of detectable transcripts in the self-incompatible B. oleracea var botrytis line 11035 indicates that SLA function is not required for the SI response.

  ) detected a single hybridizing fragment per lane at low stringency in a gel blot of restriction endonuclease digested genomic DNA from the P57Sc line, the S 2 homozygous line, and the seven B. oleracea var botrytis lines. No hybridizing fragment was detected in DNA of the class I S 3 haplotype. The SLA probe detected a 3.8-kb fragment in HindIII-digested genomic DNA from P57Sc and all seven B.

  oleracea var botrytis used in this study, five were commercial and open-pollinated varieties, either Lecerf (41865) or Automne malouin tardif (11035, 17144, 40443, and 14548), and two were non-commercial and open-pollinated varieties (24062 and 22495). Incompatibility phenotype was determined by aniline blue staining of pollinated pistils as previously described (Ruffio-Châble et al., 1997).

Figure 1 .

 1 Figure 1. Structure of the SLG/SLA gene region in the self-compatible B. oleracea line P57Sc

Figure 2 .

 2 Figure 2. Sequence similarity between the Melmoth element inserted in SLA Sc and

Figure 3 .

 3 Figure 3. SLA Sc is genetically linked to the S locus.

Figure 4 .

 4 Figure 4. Comparison of B. oleracea var botrytis lines with B. oleracea var acephala lines

Figure 5 .

 5 Figure 5. RNA gel blot analysis of SLA transcription.

(

  A) The RNA gel blot was probed with a 1260-bp EcoRV-BamHI fragment from SLA Sc (SLA probe in Figure1), and the autoradiograph was exposed for 10 days. (B) The same RNA gel blot probed with a Brassica actin cDNA probe. The autoradiograph was exposed for 6 h.Numbers at left indicate the lengths of RNA molecular markers in kilobases (kb).

Figure 6 .

 6 Figure 6. DNA blot analysis of the SLA gene.

  Taken together, the analyses of SLA, 298, 299, SLL 1 , and SLL 2 have revealed that genes exhibiting a low level of polymorphism are present at the S locus close to the high polymorphic SLG and SRK genes and that, at least in the case of SLA, localization at the S locus does not necessarily imply a role in the SI response.locus after the divergence of the different S haplotypes. SLL 1 and SLL 2 would then have spread from one S haplotype to another by homologous recombination. The fact that SLA is present in the two closely related S 2 and S Sc haplotypes but was not detected in the S 5 , S 3 , or S 3a haplotype(Boyes and Nasrallah, 1995 and this work) indicates that SLA may similarly have been inserted into the S locus during the divergence of the different S haplotypes perhaps into the common ancestor of S Sc and S 2 . We cannot, however, rule out the possibility that SLA has been deleted from the other S haplotypes during evolution or that it has been transferred from S 2 to S Sc or visa versa by a recombination event. The insertion of Melmoth into SLA presumably occurred after the divergence of the different S haplotypes in Brassica but before the divergence of B. oleracea and B. napus.

characterized two novel genes, SLL 1 and SLL 2 , located between SLG and SRK in both the 910 and A10 S haplotypes of B. napus. The DNA sequence of SLL 1 , and probably also of SLL 2 , is 100% conserved between the two S haplotypes, indicating that neither gene is involved in determining the specificity of the SI response (although one or both of these genes may play a more general role in SI).

Based on the low level of polymorphism of SLL 1 and SLL 2 compared with SLG and SRK,

[START_REF] Yu | Molecular characterization of the S locus in two self-incompatible Brassica napus lines[END_REF]

have proposed a model in which the former were inserted into the S

Table 1 .

 1 Origins and SI phenotypes of the B. oleracea var botrytis lines used in this study

			Number of	Pollen tube mean score a
	Line	Type	generations of			Phenotype b
			self-pollination	mean	min. max.
	11035	Autumn	5	0.1	0.0 0.3	SI
	24062	Autumn	14	0.3	0.3 0.3	SI
	41865	Autumn	9	0.1	0.0 0.3	SI
	17144	Autumn	6	0.9	0.0 1.7	SI
	40443	Autumn	7	0.7	0.0 1.7	SI
	14518	Winter	4	4.9	4.7 5.0	SC
	22495	Winter	9	5.4	4.0 6.8	SC

Table 2 .

 2 PCR detection of SLA in progeny issued from self-fertilization of the seven B. oleracea var botrytis plants used in this study

			Number of	Number of	Probability
	Line a	Phenotype b	progeny	PCR positive	that parent was
			tested	progeny c	homozygous
	41865	SI	29	29	99.97%
	24062	SI	17	17	99.2%
	11035	SI	17	17	99.2%
	17144	SI	10	10	94.4%
	40443	SI	15	15	98.7%
	14518	SC	13	13	97.6%
	22495	SC	16	16	99.0%

ACKNOWLEDGMENTS

We thank Marie-Angèle Grandbastien (INRA, Versailles, France) for her comments on the manuscript, Dr. Mikail E. Nasrallah (Cornell University, Ithaca, NY) for the actin probe, and Richard Blanc, Hervé Leyral, Fabienne Deguerry, Monique Estienne and Anne-Marie Thierry for technical assistance. M.P., V.R., and J.M.C. are members of the Institut National de la Recherche Agronomique, and T.G. is a member of the Centre National de la Recherche Scientifique. This work was supported jointly by the INRA and the CNRS.


Valérie Gouyer

Bastien Demouveaux

Guillaume Lacroix

Hélène Valque

Frédéric Gottrand

Jean-Luc Desseyn
email: jean-luc.desseyn@inserm.fr

Non-C-mannosylable mucin CYS domains hindered proper folding and secretion of mucin

Keywords: mucin, CYS domain, C-mannosylation Highlights

The CYS domain occurs in multiple copies in many gel-forming mucins. It is believed that CYS domains can interact with each other in a reversible manner, suggesting a key role of the domain in gel formation. This domain always contains in its amino-terminal sequence the C-mannosylation motif WXXW, but whether the CYS domain is C-mannosylated is debated, and the putative role of C-mannosylation of the domain is unclear. We prepared recombinant CYS domains of the human mucin MUC5B with (WXXW→AXXW) and without a single amino acid mutation and mini-5B mucins made of a large Ser/Thr/Pro region flanked by two CYS domains with the WXXW motif or with the mutated AXXW motif on the first, second or both CYS domains. We found that the single CYS domain and the two CYS domains of mini-5B mucin must be C-mannosylable for the efficient maturation and secretion of the recombinant molecules; otherwise, they are retained in the cell and colocalized with a resident enzyme of the endoplasmic reticulum.

Recombinant molecule analysis

Transient transfection in COS-7 cells, antibodies used and confocal microscopy are described in the Supplementary m aterials .

RT-PCR analysis of XBP1 mRNA splicing and real-time PCR analysis of CHOP mRNA

Methods to study ER stress are described in the Suppl. m aterials .

Results

Constructs

Vectors to express fusion proteins in eukaryotic cells containing the IgK signal peptide followed by the wild-type CYS domain #4 of MUC5B, mutated CYS#4 domain, or mini-5B mucins (mutated and non mutated) were prepared. The schematic representation of the insert of pC encoding the wild-type CYS domain and the nucleotide sequence are depicted in in Suppl. Fig. 1 a and b . A Thr/Ser/Ser tripeptide located downstream of the CYS sequence was then mutated into the potential N-glycosylation site Asn/Ser/Ser. To study the CYS domain in the context of the mucin structure, the CYS domain must be linked to O-glycosylated sequences. In this context, the CYS sequence is flanked by two short peptides that are predicted to be O-glycosylated according to the NetOGlyc 3.1 Server [START_REF] Julenius | Prediction, conservation analysis, and structural characterization of mammalian mucin-type Oglycosylation sites[END_REF] (Suppl. Fig. 1 b ). The alignment of the N-terminal aa sequences of CYS domains from MUC2, MUC5B, MUC5AC, and mouse Muc5b using WebLogo (Suppl. Fig. 1 c ) point to strong sequence conservation of the C-mannosylation sequence, Cys residues, and other aa residues.

The recombinant CYS domain is secreted into the cell culture medium as N-and O-glycosylated molecules

COS-7 cells were transfected transiently with pC and the cell culture media and cell lysates were analyzed by Western blotting. pC was secreted as expected as different glycoproteins of molecular weights ~42kDa (Suppl. Fig. 2 a ). The calculated molecular weight of the secreted naked pC was 16kDa, indicating posttransductional modification of the domain that is compatible with glycosylation. pC was also detected in cell lysates as one major protein with molecular mass of 22kDa, which corresponds to the calculated weight of the N-glycosylated form. This was confirmed by tunicamycin treatment of pC-transfected COS-7 cells showing a band of 16kDa corresponding to the non Nglycosylated pC molecule (Suppl. Fig. 2 a ). Confocal microscopy analysis showed that pC localized at the cell membrane surface (Suppl. Fig. 2 b ). Similar images were obtained without and under a permeable condition confirming the localization of pC outside of the cell (Suppl. Fig. 2 c ). Altogether, these data demonstrated the secretion of pC.

The CYS domain must be C-mannosylable for proper secretion of the domain

To assess if CYS domain for which its C-mannosylation site is abolished is still secreted, a vector expressing mutated WXXW→AXXW CYS domain (pC*) was prepared. Its schematic structure is depicted in Fig. 1 a . The mutation of the first Trp in the C-mannosylation site prevented its secretion into the cell culture medium as shown by Western blot (Fig. 1 b ) and in agreement with others [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF]. The non-Cmannosylable pC* was retained in the cell as two proteins with molecular mass of 16 and 22kDa (Fig. 1 c ); these probably correspond to the naked peptide and the N-glycosylated peptide, respectively. Confocal microscopy confirmed that pC was secreted while pC* was not with a perinuclear localization (Fig. 1 d ). Nonpermeabilized cells transfected with pC* did not stain with the specific antibody (Ab; Fig. 1 e ). Colocalization experiments by immunofluorescence showed that pC*, but not pC, colocalized with calnexin (Fig. 1 f ), a resident lectin of the ER. In contrast, both pC* and pC do not colocalized with ST3GalI, a resident glycosyltransferase of the trans-Golgi network (Fig. 1 g ). Taken together, these findings demonstrate that the mutation WXXW→AXXW blocks the CYS domain in the ER, and prevents its transport into the Golgi apparatus. The ER localization of the mutated molecules prompted us to look for possible ER stress. Accumulation of unfolded or misfolded proteins in the ER triggers the unfolded protein response (UPR), also called ER stress. This process is marked by the induction of protein expression or splicing activation of several ER stress-sensor proteins [START_REF] Yoshida | pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response[END_REF]. We looked for activation of two among the three UPR pathways known to be activated after ER stress [START_REF] Wang | Protein misfolding in the endoplasmic reticulum as a conduit to human disease[END_REF]. The first one is the inositol-requiring protein 1a (IRE1a)-X-box-binding protein 1 (XBP1) pathway where a specific endoribonuclease activity cleaves a small nucleotide segment of XBP1 mRNA, which we examined using reverse transcript-polymerase chain reaction in COS-7 cells transiently transfected with pC and pC*. The cleaved form of XBP1 mRNA was found at a higher levels in COS-7 cells producing pC* than in cells transfected producing pC, indicating the activation of ER stress in cells expressing pC* (Suppl. Fig. 3a andb). The second UPR pathway we looked at is the activating transcription factor (ATF)6a, which activates the transcription of CCAAT/enhancer-binding protein C/EBP homologous protein (CHOP) [START_REF] Zinszner | CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum[END_REF].CHOP mRNA expression was measured by quantitative PCR. Consistent with an activated unfolded protein response for pC*, CHOP mRNA expression was found to be 4-fold higher (P=0.02) in COS-7 cells transfected with pC* compared with COS-7 cells transfected with pC (Suppl. Fig. 3 c ).

The mini-5B mucin is secreted into the cell culture medium in two major glycoforms

Because the CYS domain is never found as a single copy in mucin, we constructed an expression plasmid encoding a secreted fusion protein containing two MUC5B CYSdomains flanking a tandem array of 11 irregular repeats from MUC5B followed by a Rend domain (111 aa polypeptide enriched in Ser/Thr/Pro), which we named pCtrC. This recombinant mini-5B/pCtrC (Suppl. Fig. 4 a ) is representative of a mini-MUC5B that lacks the globular amino-and carboxy-terminal regions necessary for the mucin polymerization. The deduced aa sequence of the Ser/Thr/Pro/ region (Suppl. COS-7 cells were transiently transfected with the pCtrC construct, and the cell culture medium and cell lysate were analyzed as before by Western blotting. Mini-5B was secreted in at least two major glycoforms of high molecular weight (>160kDa) (Fig. 2 a ). Mini-5B was also found in an immature form in cell lysates with an apparent molecular weight of ~98kDa corresponding likely to the nonglycosylated form (Fig. 2 a ). The analysis of the secreted proteins with different lectins showed that the two main bands exhibited different glycosylation patterns: the higher molecular weight glycoform was sialylated as shown by the MAA lectin (Fig. 2 b ) while the two bands correspond to molecules carrying mucin type carbohydrate T-antigens as shown by the PNA lectin. The secretion of mini-5B was confirmed by confocal microscopy in transiently transfected cells for both permeabilized and nonpermeabilized cells (Fig. 2 c andd ).

All CYS domains must be C-mannosylable for efficient mucin secretion

To further evaluate whether the two CYS domains must be C-mannosylable for the mini-mucin secretion, we prepared different mutants of pCtrC, which were mutated on the C-mannosylation site of the first CYS domain (pC*trC) or on the C-mannosylation site of the second CYS domain (pCtrC*) or on both C-mannosylation sites (pC*trC*) (Fig. 3 a ). Plasmid vectors were transiently transfected into COS-7 cells. Western blotting was first used to analyze the cell culture media and cell lysates. A higher volume/concentration of supernatant from COS-7 cells transfected with constructs encoding all mutated mini-5B, which was estimated to be at least five times higher than the wild-type mini-5B, was loaded in order to confirm that COS-7 cells were efficiently transfected. pCtrC was secreted with a MM >160kDa as previously shown (Fig. 2 a ). A thin band at >160kDa and a major product at ~98kDa were detected for pC*trC and pCtrC* and corresponds to an immature nonglycosylated mini-5B (Fig. 3 b ). The mutation of both C-mannosylation sites (pC*trC*) fully abolished mucin production (Fig. 3 b ). All mutant products were found as an immature form of ~98kDa in cell lysates (Fig. 3 c ) suggesting that the 98kDa band observed in media corresponds to released mini-mucin by cell death. The secretion of pCtrC but not mutated mini-mucins was next confirmed by confocal microscopy, which showed that the pCtrC product localized at the external cell surface membrane (Fig. 3 d ). However, all mutated mini-5B exhibited mainly a perinuclear localization (Fig. 3 d ). Absence of secretion of pC*trC was confirmed using nonpermeabilized transfected cells (Fig. 3 e , compared with pCtrC in Fig. 2 d ). To determine where the mutated mni-5B localized within the cell, immunofluorescence experiments were conducted using antibodies directed against RE and Golgi resident proteins. pCtrC did not co-localized with calnexin while each single and the double mutated mini-5B colocalized with calnexin in the ER (Suppl. Fig. 5 a ) but not with ST3GalI in the Golgi apparatus as shown for pC*trC and pC*trC* (Suppl. Fig. 5 b ).

Discussion

The CYS domain is the best-conserved domain in gelforming mucins, suggesting that it plays a key role [5 ,7 ,21 ,22] . Its sequence seems to have undergone concerted evolution with a high selective pressure to conserve cysteine residues and few other aa, particularly on the C-mannosylation consensus sequence located in the amino-terminal part of the domain [5].

Conclusions from published works on the Cmannosylation of mucin CYS domains are conflicting. A report using recombinant CYS domains produced by the fibroblastic cell line CHO-K1 Lec 3.2.8, which is deficient in the enzyme GnT-I transferase and the UDP-galactose transporter [START_REF] Oelmann | Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDPgalactose and CMP-sialic acid transporters[END_REF], suggested that the CYS domain is not C-mannosylated [START_REF] Ambort | Function of the CysD domain of the gel-forming MUC2 mucin[END_REF]. By contrast, other data suggest that all mucin CYS domains are probably C-mannosylated. First, the C-mannosylation consensus sequence (WXXW/F/Y) is found in all CYS domains reported previously (135 copies in seven species) as shown in Suppl. Fig. 6 according to the consensus sequences of the amino-terminal parts of CYS domains found in all species. In addition, all secreted molecules that have a WXXW/Y/F sequence conserved between species, such as properdin, thrombospondin, and F-spondin, have been shown to be C-mannosylated (for review, see [1]). The extreme selective pressure upon the C-mannosylation sequence, not forgetting that to the two tryptophan residues correspond a single TGG codon, indicates that the sequence has likely been conserved for the C-mannosylation event. Second, at least two MUC5AC (#1 and #5) and three MUC5B CYS domains (#1-3) are C-mannosylated according to a report using recombinant CYS domains from these two mucins [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF], although the conclusion of the authors relied on the characterization of mutated recombinant CYS domains without any direct biochemical proof concerning the C-mannosylation. Mass spectrometry analysis of MUC5B and MUC5AC isolated from human tracheobronchial epithelial (HTBE) cells and Calu-3 cells (airway epithelial cells), respectively, suggested that the first three CYS domains of MUC5B are likely not C-mannosylated but the authors could not rule out the presence of the modification at low level and identification of C-mannose in other CYS domains of MUC5B and in CYS domains of MUC5AC was not possible with the method used [START_REF] Cao | Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study[END_REF]. Third, MUC2 contains three sequences that may be C-mannosylated: one in each of its two CYS domains (WSDW and WTGW in MUC2 CYS#1 and #2, respectively) and a third (WGNF) in its amino-terminal region. Gas chromatography/MS analysis of colonic mucins containing mainly or only MUC2 showed that there are three C-mannosylation events for one MUC2 molecule, supporting the concept that the three C-mannosylation sequences of MUC2 are C-mannosylated [START_REF] Zanetta | Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins[END_REF]. This is consistent with our conclusion from our observations of mini-5B that the two CYS domains must be C-mannosylable for the secretion of the mini-mucin supporting that all CYS domains are likely C-mannosylated for the proper secretion of the mucin. The inconsistency of conclusions drawn on independent studies about the C-mannosylation of mucins might suggest that, depending on the biological sample used, C-mannosylation of the CYS domain may be required. Coexistence of both C-mannosylated and un-C-mannosylated molecule has already been reported [START_REF] Goto | C-mannosylation of human hyaluronidase 1: possible roles for secretion and enzymatic activity[END_REF].

Unfortunately, our many attempts to demonstrate using mass spectrometry that our recombinant molecules are or not C-mannosylated were unsuccessfull. The main hurdles are the low production of recombinant molecules by transient expression, the low efficiency of purification of the molecules, the predicted high hydrophobicity of the CYS domain and the N-and O-glycosylation of recombinant molecules that greatly hinder the mass spectrometry analysis.

Obtaining a full recombinant gel-forming mucin is a difficult task because of the extremely large size of mucin mRNA (>15kb). To study the CYS domain in the context of the Ser/Thr/Pro region, we designed a construct encoding a full Ser/Thr/Pro region of MUC5B (440aa) flanked by two CYS domains (110aa). A strong point of all our constructs was the introduction of an N-glycosylation site because gelforming mucins are N-glycosylated in their C-terminal region [START_REF] Thornton | Structure and function of the polymeric mucins in airways mucus[END_REF] and several potential N-glycosylation sites are conserved in mucins [START_REF] Desseyn | Genomic organization of the 3' region of the human mucin gene MUC5B[END_REF]. N-glycans are probably required to maintain a slow folding rate for mucins, which allows proper maturation. N-glycosylation is also important for correct protein folding, quality control, sorting, and transport [29 ,30] . In the first published work on C-mannosylation of mucin, recombinant His-tagged CYS domains of MUC5B and MUC5AC were visualized only after cell radiolabeling followed by absorption and purification using the poly-His tag [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF] indicating a poor efficiency of production/secretion using COS-7 cells, CHO-Lec31.1 and CHO-K1 cells. Furthermore, when these constructs were fused to the green fluorescent protein sequence, recombinant molecules were found mainly in the Golgi complex. In contrast, our wild-type CYS molecule and mini-5B were secreted exclusively. Taken together, these results show that the tools we designed have all the features required to study the biological role of the CYS domain and the mini-5B mucin. In this line, we reported that mini-5B promotes MCF7 cell proliferation and invasion in vitro and tumor growth and tumor cell dissemination in vivo using stable MCF7 transfected clones [START_REF] Valque | MUC5B Leads to Aggressive Behavior of Breast Cancer MCF7 Cells[END_REF].

C-mannose may play a structural role in stabilizing C-mannosylated molecules and in the proper transport from the ER to the Golgi apparatus via putative lectin recognition [1 ,8] . Our results demonstrate that recombinant mutated molecules are blocked in the ER. This result supports a key role in the quality control system of nascent protein in the ER. This suggests that mutated recombinant molecules tend to misfold and then to fail the quality control [START_REF] Trombetta | Quality Control and Protein Folding in the Secretory Pathway[END_REF], which induces ER stress. If our CYS domains are Cmannosylated, then it is possible that C-mannose stabilizes the CYS domain. It is also possible that the C-mannose, as suggested for N-glycans [START_REF] Ruddock | N-glycan processing in ER quality control[END_REF], may contribute substantially to increasing the hydrophilicity of the highly hydrophobic CYS domain, which then inhibits aggregation. An attractive hypothesis is that the C-mannose participates actively to the mucin intra-and/or inter-chain bridges as suggested for the C-mannosylation of the receptor of IL-21 [START_REF] Hamming | Crystal structure of interleukin-21 receptor (IL-21R) bound to IL-21 reveals that sugar chain interacting with WSXWS motif is integral part of IL-21R[END_REF].

Supplementary Materials

Constructs

All vector construction PCRs were performed using Pfu Turbo (Stratagene/Agilent Technologies, Massy, France). Primers included appropriate restriction cleavage sites to allow further insertion of the PCR products into the expression vectors. PCR products were cloned into the pMosBlue Blunt vector (Amersham biosciece/GE Healthcare Velizy-Villacoublay, France). Cloned PCR fragments were excised from pMosBlue Blunt with the appropriate restriction enzymes (New England Biolabs, Evry, France) to be subcloned. CYS domain-expressing vectors pC and pC* (wild-type CYS domain and mutated CYS domain on the C-mannosylation site, respectively) and the mini-mucin mini-5B called hereafter pCtrC (wildtype and mutated pC*trC, pCtrC*, and pC*trC*) were obtained using the following strategy. Briefly, the cytomegalovirus immediate early promoter of the pcDNA3.1 Neo+ (Invitrogen/Life technologies, Villebon sur Yvette, France) carried by the 940bp genomic sequence BglII-EcoRI was replaced by a 845bp PCR-amplified EF1α-HTLV composite promoter from pMG (InvivoGen, Toulouse, France) flanked by the two unique BamHI and EcoRI restriction sites, producing the expressing vector pcMG. The BEN2 genomic clone containing the large central exon of MUC5B [1] was used to amplify the two intronless fragments coding either for the CYS#4 domain alone (fragment C) or for CYS#4 followed by a long Ser/Thr/Pro-rich region made of 11 irregular TRs of 29 amino acid (aa) residues and a Rend domain of 111 aa residues (fragment Ctr). The CYS#4 of MUC5B was chosen because specific antibodies against a peptide sequence belonging to this domain (see Antibodies section below) were available. We mutated the sequence encoding the tripeptide TSS of the CYS sequence to the N-glycosylation site NSS using the QuickChange Site-Directed Mutagenesis protocol from Stratagene using the two oligonucleotide sequences 5'-CCAGCACCCCGGCCAACAGCTCTACGGCCAC-3' (forward) and 5'-TGGCCGTAGAGCTGTTGGCCGGGGTGCTGGG-3' (reverse). The fragment carrying the CYS sequence was subcloned into the pSecTagB vector (Invitrogen) in frame with the IgK leader chain. The IgK-CYS sequence was then subcloned into the pcMG vector (expression vectors pC and pCH6, respectively).

The C-mannosylation site (WSEW) of pC was changed to ASEW (vector pC*) using two oligonuceotides with the following sequence: 5'-TGAGCCCCAGTGTGCCGCGTCAGAGTGGCT-3' (forward) and 5'-AGCCACTCTGACGCGGCACACTGGGGCTCA-3' (reverse). The amplified genomic fragment Ctr was also mutated on the C-mannosylation site (WXXW→AXXW; fragment C*tr). The wild-type and mutated fragments were subcloned in frame between the signal peptide and the CYS sequence from the pC and the pC* vectors, producing the four expression vectors pCtrC, pC*trC, pCtrC*, and pC*trC*.

Antibodies

Polyclonal antibodies (pAb) against human calnexin (H70) and β-actin (ab8227) were purchased from Santa Cruz (Santa Cruz Biotechnologies, Heidelberg, Germany) and abcam (Cambridge, UK), respectively. Mouse monoclonal antibodies (mAbs) against MUC5B (Eu1 and Eu2) and ST3GalI (4B10) were generous gifts from D. Swallow and K. Rousseau [2] from the European Consortium (Concerted Action contract number BMH4-CT98-3222) and U. Mandel [3] (School of Dentistry, Copenhagen, Denmark), respectively. The rabbit anti-MUC5B pAb LUM5B-2 [4] was a gift from I. Carlstedt (Lund University, Sweden).

Cell culture and transient transfection COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 200mM L-glutamine, and 100U penicillin/streptomycin (Invitrogen). Transient transfection of COS-7 cells was performed using Effectene (Qiagen, Courtaboeuf, France). Cells were seeded in standard medium in 10mm Petri dishes. Cells were transfected using 2µg of plasmid according to the manufacturer's instructions when cells reached 70-80% confluency. Twenty-four hours after transfection, cells were cultured in serum-free medium containing 0.01% bovine serum albumin. The cell culture media and cells were collected 48 h after transfection, and the cell culture media were concentrated in a Centricon-3 concentrator (Amicon, Bedford, MA). For tunicamycin treatment, cells were transfected with the pC expression plasmid, and 24 h later, the cells were treated with 10µg/mL tunicamycin for 1 h before harvest.

Confocal microscopy

Confocal microscopy was performed on transiently transfected COS-7 cells. The cells were fixed in 4% paraformaldehyde for 20 min, quenched for 30 min with 50mM NH4Cl in phosphate-buffered saline (PBS), and permeabilized with 0.2% saponin in PBS for 20 min. The saturation step was performed for 30 min in PBS containing 1% bovine serum albumin and 0.2% saponin. To detect MUC5B, ST3GalI, and calnexin, mAbs Eu1 and Eu2 (diluted 1/600), 4B10 (1/4), and rabbit pAb H-70 (1/200), respectively, were added overnight. LUM5B was used for double labeling of MUC5B/calnexin and MUC5B/ST3GalI. For single labeling, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse Ab for 90 min, and the nuclei were counterstained with propidium iodide or Hoechst 33258. For double labeling, fluorescein isothiocyanate-conjugated goat anti-rabbit Ab and Texas-Red conjugated goat antimouse Ab (Jackson Immunoresearch, Interchim, Montluçon, France) were used. In order to confirm the secretion and the localization at the membrane of the recominant molecules, immunolabeling was performed as described above but without permeabilization, i.e. in absence of saponin. Confocal microscopy was performed using a DMIRBE microscope (model TCS-NT; Leica) with a 40x 1.32 Plan-Apochromat oil-immersion objective lens. Acquisition was performed using PowerScan software (Leica) and processed with Adobe Photoshop CS.

Protein detection by Western blot and lectin blot analysis

The cell culture medium was collected (5mL) and concentrated to 200µL by centrifugation in a Centricon-3 tube (Amicon Millipore, Molsheim, France). Cells were washed five times with PBS and resuspended in 200µL PBS containing 0.2mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. The cells were freeze-thawed and sonicated, and the protein concentration was measured using the BCA protein assay (Pierce Biotechnology, Rockford, IL). Protein lysates (usually 30 µg) and supernatants (usually 20 µL) were loaded onto an 8% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a vertical gel apparatus (Bio-rad, Marnes-la-Coquette, France), electrophoresed, and then transferred to a nitrocellulose membrane. To analyze the mini-mucins, the supernatant of mutated mini-5B was concentrated to be at least five times the concentration of the wild-type mini-5B. The membrane was blocked with 5% powdered milk in PBS/0.1% Tween 20 (TPBS) overnight at 4°C, washed, and probed with mAb against MUC5B diluted 1:600 in TPBS. After washing, the membrane was incubated for 45 min with horseradish peroxidase-conjugated goat anti-mAb (Santa Cruz Biotechnology) diluted 1:2000 in TPBS. The membrane was then washed for 30 min in TPBS, and an ECL™-Plus chemiluminescent detection kit (Amersham Biosciences, GE Healthcare) was used for visualization (5 min). To normalize protein expression the membrane with cell lysates was then probed with pAb against β-actin diluted 1:3000 in TPBS. For lectin blots, after blotting, the nitrocellulose membrane was treated for 2 h with 2% polyvinylpyrrolidone K 30 in 10mM Tris-HCl, pH 7.4, containing 0.15M NaCl (TBS). The membrane was incubated in digoxigenin-labeled MAA lectins, which recognize the oligosaccharide species Neu5Acα2-3Gal-R or peanut Arachis hypogaea (PNA), which recognizes Galα1-3GalNAc-R (Antigen-T) at a concentration of 5 μg/mL in TBS. The nitrocellulose membrane was incubated for 1 h with alkaline phosphatase-labeled antidigoxigenin Fab fragment (1μg/mL in TBS). The labeled glycoproteins were revealed by 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate staining.

RT-PCR analysis of XBP1 mRNA splicing and real-time PCR analysis of CHOP mRNA

Total RNA was extracted and reverse transcribed as before [5]. To detect spliced XBP1 mRNA, the PCR conditions were 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 30 cycles using the primer pair 5'-CCTTGTAGTTGAGAACCAGG-3' and 5'-GGGGCTTGGTATATATGTGG-3', which yielded a 424bp unspliced XBP1 fragment and a 442bp spliced XBP1 fragment. G3PDH amplification was used as endogenous control (Clontech, Saint-Germain-en-Laye, France). PCR amplifications were analyzed by electrophoretic migration on an 8% acrylamide gel and stained with ethidium bromide. Band intensity was analyzed using ImageJ (NIH, Bethesda, MD, USA) and the intensity of the spliced-form was normalized to the intensity of the unspliced form. For quantitative PCR analysis of CHOP mRNA, primer and TaqMan probe sequences were selected using the Primer3 Output program (MIT) (Primer3 freeware v0.4.0) with the Macaca CHOP cDNA sequence (Macaca mulatta gene ENSMMUG00000011286 (DDIT3)). The specific primers and probe for CHOP were as follows: macacaCHOP forward primer, 5'-GCCAAAATCAGAGCTGGAAC-3' belonging to exon 1 and macacaCHOP reverse primer, 5'-GCTTTCAGGTGTGGTGATGT-3' belonging to exon 2; and macacaCHOP probe, 5'-GGAGAGAGTGTTCAAGAAGGAAGTGTA-3' overlapping the exon 1-exon 2 junction to avoid amplifying contaminating genomic DNA. The 18s rRNA was chosen as the internal positive control. PCR analysis was performed as described previously. All samples were measured in triplicate. The cycle threshold values of all samples were measured using the ABI Prism 7700 sequence detector system (Applied Biosystems), and results are expressed using the 2 -ΔΔCt method relative to the changes in gene transcription. For each sample, the ΔΔCt value was obtained by subtracting the ΔCt value for 18s rRNA from that of the CHOP mRNA, and the ΔΔCt was obtained by subtracting the ΔCt value of the pC-transfected COS-7 cells from that of the pC mutant-transfected COS-7 cells. Data are expressed as mean ± standard deviations of triplicate determination. Statistical significance was evaluated using the Student's t-test for unpaired comparison. Difference with p < 0.05 was considered to be statistically significant.

Supplementary References

Figure 1 .

 1 Figure 1. Requirement of the C-mannosylation sequence for the proper folding and secretion of the CYS domain. (a) Schematic representation of the sequences of interest of pC*. ATG initiating Met, C-mannosylation sequence (W) and two AATAAA sequences are indicated. S, secretion peptide; ORF, open reading frame; UTR, untranslated region; YAb, Ab-recognition peptide sequence of MUC5B. N, Nglycosylation site. Mutated aa residue is indicated in red (W→A). (b, c) Western blot analysis in of cell culture media of COS-7 cells transiently transfected with pC and pC*. b-media, pC* was not secreted. ccell lysates, pC was found in two forms. (d-g) Transfected cells were studied by confocal microscopy. The nuclei were counterstained by propidium iodide (red) or with Hoechst 33258 (blue). (d) Immunostaining with anti-MUC5B antibody (green) showing that pC* exhibited likely perinuclear localization. (e) Confocal analysis of transiently transfected COS-7 cells with pC* and analyzed with anti-MUC5B Ab (green) without (top) or with (below) permeabilization confirming that pC* is retained within cells. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50µm. (f) Colocalization experiments using Abs directed against calnexin (ER marker) showing that the mutant construct pC* was blocked in the ER. (g) pC was secreted and localized to the cell membrane while pC* was retained in the cells and do not colocalized with ST3GalI in the Golgi apparatus. d,f,g: scale bar, 10 µm. e, scale bar, 50µm.

  Fig. 4 b ) and aa composition of the Ser/Thr/Pro region and the full pCtrC are given (Suppl. Fig. 4 c and d ) outlining the high content of Ser and Thr in the Ser/Thr/Pro region and the high content of Cys residues in the CYS domain. The pCtrC mini-5B construct depicted in Suppl. Fig. 4 e .

Figure 2 .

 2 Figure 2. Secretion of mini-5B (pCtrC) as a highly O-glycosylated molecule. (a) Western blot analysis showing that pCtrC was secreted in at least two forms with Mr >160kDa. pCtrC was found with an Mr ~98kDa in cell lysates. (b) pCtrC analyzed by Western blotting using PNA and MAA lectins showing that pCtrC was secreted as two main glycoforms, one of which was sialylated. (c) Confocal microscopy showing that pCtrC (green) is secreted. Scale bar 10 µm. (d) Confocal analysis of pCtrC (green) without (left panel) or with (right panel) permeabilization confirming that pCtrC is secreted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50µm.

Figure 3 .

 3 Figure 3. Both CYS domains of pCtrC must be C-mannosylable for the proper secretion of the recombinant mucin. (a) Schematic representation of the sequences of interest of mutated mini-mucins. Western blot analysis of (b) cell culture media and (c) cell lysates of COS-7 cells transiently transfected with the expression plasmids pCtrC, pC*trC*, pC*trC, or pCtrC*. In b, the 3 mini-5B with mutation were overloaded on purpose. (d,e) Confocal microscopy of COS-7 cells transiently transfected with mini-5B constructs. (d) Mutated mini-5B showed perinuclear localization. The cells were immunostained with anti-MUC5B Ab (green), and the nuclei were counterstained with propidium iodide (red). Scale bars, 10 µm. (e) pC*trC (green) was confirmed to be not secreted as revealed without (left panel) permeabilization of COS-7 cells. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50µm.

Acknowledgements

We thank D. Swallow and K. Rousseau for the MUC5B monclonal Ab, U. Mandel for the ST3GalI Ab and I. Carlstedt for the anti-MUC5B pAb LUM5B-2.

Funding

BD is the recipient of a fellowship from the Région Hautsde-France and the Centre Hospitalo-Universitaire de Lille. GL is the recipient of a fellowship from the Université de Lille. This work was supported by a the Région Hauts-de-France [grant ARCIr Volet Dynamique 1400324].

Author's contributions JLD designed the constructs. HV, BD, GL and VG performed in vitro experiments. VG and JLD wrote the manuscript with the critical reading of FG. All authors approved the final manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.138. (a) Confocal analysis of transiently transfected COS-7 cells with pCtrC, pC*trC, pCtrC* and pC*trC* using an anti-MUC5B (green) and anti-calnexin antibodies (in red). All mutated mini-5B were retained within the cell and colocalized (yellow) with calnexin in the ER while the control wild-type mini-5B pCtrC was secreted by cells. (b) Pictures illustrating that mini-5B (in green) with a WXXW→AXXW mutation in its first CYS domain or the mutation within its two CYS domains were retained in the cells but did not colocalized with ST3GalI (red) in the Golgi apparatus. Scale bars, 10µm.

Suppl. Figure 6. CYS domains containing the C-mannosylation sequence WXXW in their amino-terminal region. CYS sequences were used to generate a WebLogo for each species. The number of CYS domains found in each species and the number of genes (human and one tunicate gene) or putative genes encoding multiple copies of CYS domains are indicated. Two CYS sequences from oikosine-1 secreted by a tunicate contained the WXXY and WXXF peptide sequences that could be C-mannosylated. Two CYS sequences from the zebrafish contained the WXXP/A sequences that could not be C-mannosylated.