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Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function*

Open AccessPublished:August 21, 2010DOI:https://doi.org/10.1074/jbc.M110.109553
      Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

      Introduction

      Chondroitin sulfate (CS)
      The abbreviations used are: CS
      chondroitin sulfate
      CH
      chondroitin
      CSA-8
      octasaccharide of chondroitin sulfate A
      CSC-8 and -9
      octasaccharide and nanosaccharide of chondroitin sulfate C
      CSS
      chondroitin sulfate synthase
      ChSy
      chondroitin synthase-1
      ER
      endoplasmic reticulum
      GalNAcT
      N-acetylgalactosaminyltransferase
      GlcUA
      d-glucuronic acid
      GlcAT
      glucuronyltransferase
      GAG
      glycosaminoglycan
      MEF
      mouse embryonic fibroblast
      ChPF
      chondroitin polymerizing factor
      Mfng
      mouse fringe.
      is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine (GalNAc) and d-glucuronic acid (GlcUA) residues, modified with sulfated residues at various positions (
      • Kjellén L.
      • Lindahl U.
      ,
      • Esko J.D.
      • Selleck S.B.
      ,
      • Prydz K.
      • Dalen K.T.
      ,
      • Sugahara K.
      • Kitagawa H.
      ). CS chains exhibit structural diversity in chain length and sulfation patterns, providing specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division (
      • Perrimon N.
      • Bernfield M.
      ,
      • Sugahara K.
      • Mikami T.
      • Uyama T.
      • Mizuguchi S.
      • Nomura K.
      • Kitagawa H.
      ,
      • Deepa S.S.
      • Yamada S.
      • Zako M.
      • Goldberger O.
      • Sugahara K.
      ,
      • Bao X.
      • Muramatsu T.
      • Sugahara K.
      ,
      • Oohira A.
      • Matsui F.
      • Tokita Y.
      • Yamauchi S.
      • Aono S.
      ,
      • Morgenstern D.A.
      • Asher R.A.
      • Fawcett J.W.
      ).
      CS biosynthesis is initiated by transfer of GalNAc to the linkage region of a GlcAβ1–3Galβ1–3Galβ1–4Xyl tetrasaccharide primer that is attached to a serine residue of a core protein. Then chain polymerization takes place by the alternative addition of GalNAc and GlcUA residues. The enzymatic activities that catalyze CS initiation and polymerization processes are designated glycosyltransferase-I and -II activities, respectively (
      • Sugahara K.
      • Kitagawa H.
      ). To date six glycosyltransferases for chondroitin synthesis have been identified: chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1), chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor (ChPF), chondroitin sulfate synthase-3 (CSS3)/chondroitin synthase-2 (ChSy-2), chondroitin sulfate glucuronyltransferase/chondroitin synthase-3 (ChSy-3), and chondroitin N-acetylgalactosaminyltransferase-1 and -2 (
      • Yada T.
      • Gotoh M.
      • Sato T.
      • Shionyu M.
      • Go M.
      • Kaseyama H.
      • Iwasaki H.
      • Kikuchi N.
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      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Narimatsu H.
      • Kimata K.
      ,
      • Gotoh M.
      • Yada T.
      • Sato T.
      • Akashima T.
      • Iwasaki H.
      • Mochizuki H.
      • Inaba N.
      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Kimata K.
      • Narimatsu H.
      ,
      • Kitagawa H.
      • Uyama T.
      • Sugahara K.
      ,
      • Kitagawa H.
      • Izumikawa T.
      • Uyama T.
      • Sugahara K.
      ,
      • Izumikawa T.
      • Koike T.
      • Shiozawa S.
      • Sugahara K.
      • Tamura J.
      • Kitagawa H.
      ,
      • Sato T.
      • Gotoh M.
      • Kiyohara K.
      • Akashima T.
      • Iwasaki H.
      • Kameyama A.
      • Mochizuki H.
      • Yada T.
      • Inaba N.
      • Togayachi A.
      • Kudo T.
      • Asada M.
      • Watanabe H.
      • Imamura T.
      • Kimata K.
      • Narimatsu H.
      ,
      • Uyama T.
      • Kitagawa H.
      • Tanaka J.
      • Tamura J.
      • Ogawa T.
      • Sugahara K.
      ). All contain an N-terminal transmembrane domain; thus, they are type II-membrane proteins. CSS1, CSS2, and CSS3 contain two glycosyltransferase domains, β-3 domain at the N-terminal region and β-4 domain at the C-terminal region and exhibit dual enzymatic activities of N-acetylgalactosaminyltransferase-II (GalNAcT-II) and glucuronyltransferase-II (GlcAT-II). Chondroitin sulfate glucuronyltransferase, similarly containing two glycosyltransferase domains, shows only GlcAT-I activity (
      • Gotoh M.
      • Yada T.
      • Sato T.
      • Akashima T.
      • Iwasaki H.
      • Mochizuki H.
      • Inaba N.
      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Kimata K.
      • Narimatsu H.
      ), although another report also indicated GalNAcT activity (
      • Izumikawa T.
      • Koike T.
      • Shiozawa S.
      • Sugahara K.
      • Tamura J.
      • Kitagawa H.
      ). Chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2 exhibit both GalNAcT-I and GalNAcT-II activities responsible for chain initiation and polymerization, respectively. Although CSS1, CSS2, and CSS3 show both GlcAT and GalNAcT activities, none of these enzymes show chain polymerization individually. Characterization of these enzymes has revealed that samples obtained from cells co-expressed with a combination of two dual enzymes has polymerizing activity, but a mixture of the two does not (
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ). These observations suggest that a complex of two glycosyltransferases is the core machinery for CS polymerization and that it can only be formed in the cell.
      Because coexpression of dual enzymes has been shown necessary for CS chain polymerization (
      • Izumikawa T.
      • Koike T.
      • Shiozawa S.
      • Sugahara K.
      • Tamura J.
      • Kitagawa H.
      ,
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ), the other two enzymes may be redundant, or different pairs may exert a distinct function. In addition, co-expression of different pairs causes differences in chain length polymerized (
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ). However, the mechanism of how the partnership causes the chain length remains to be understood. As various factors including sulfation levels and patterns have been shown to affect CS chain length (
      • Uyama T.
      • Ishida M.
      • Izumikawa T.
      • Trybala E.
      • Tufaro F.
      • Bergström T.
      • Sugahara K.
      • Kitagawa H.
      ,
      • Klüppel M.
      • Wight T.N.
      • Chan C.
      • Hinek A.
      • Wrana J.L.
      ), the differences in CS chain length observed by in vitro assay systems may not recapitulate in vivo CS biosynthesis.
      CSS1, CSS2, CSS3, and chondroitin sulfate glucuronyltransferase are expressed ubiquitously rather than in a tissue-specific manner, although there are some exceptions. Among these enzymes, CSS1 exhibits the highest expression level and GlcAT and GalNAcT activities followed by CSS2 (
      • Kitagawa H.
      • Uyama T.
      • Sugahara K.
      ,
      • Kitagawa H.
      • Izumikawa T.
      • Uyama T.
      • Sugahara K.
      ). In contrast, the expression levels of CSS3 are considerably low (
      • Yada T.
      • Sato T.
      • Kaseyama H.
      • Gotoh M.
      • Iwasaki H.
      • Kikuchi N.
      • Kwon Y.D.
      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Narimatsu H.
      • Kimata K.
      ). These observations strongly suggest that the complex of CSS1 and CSS2 plays the major role in CS chain polymerization in vivo. However, the molecular mechanisms of complex formation and its CS chain polymerization have not been elucidated.
      Fortuitously, we found an alternative splice variant of mouse CSS2 in the gene data base at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD). The splice variant (GenBankTM accession number NM_001001565) is supposed to lack the transmembrane, contrasting to the original/authentic CSS2 (GenBankTM accession number NM_001001566). In this study we investigated the roles of CSS2 by comparing the original enzyme CSS2A with the splice variant CSS2B. Both CSS2A and CSS2B were indeed expressed in various tissues and were localized in both the endoplasmic reticulum (ER) and the Golgi apparatus. Pulldown assays revealed homogenous and heterogeneous interaction among CSS2A, CSS2B, and CSS1/ChSy-1, suggesting that they form a complex of multimers. A series of in vitro glycosyltransferase assays demonstrated a reduced GlcAT activity in CSS2B that was supported by molecular modeling. CSS2B co-expressed with CSS1 exhibited no polymerizing activity, in contrast to CSS2A co-expressed with CSS1. Moreover, analysis of CS biosynthesis revealed inhibition by CSS2B. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight into the mechanisms of CS biosynthesis by CSS1 and CSS2.

      DISCUSSION

      In this study we have found cDNA for a splice variant of mouse CSS2/ChPF and have shown for the first time that both the original CSS2 and the splice variant, designated CSS2A and CSS2B, respectively, are synthesized as proteins. Compared with CSS2A, CSS2B lacks the N-terminal region of 162 amino acid residues including the N-terminal transmembrane domain and a putative N-linked oligosaccharide attachment site. Their characterization revealed several points as follows. 1) Whereas CSS2B exhibits a similar level of GalNAcT activity to that of CSS2A, it exhibits ∼26% GlcAT activity that of CSS2A; 2) even without the N-terminal transmembrane domain, a large proportion of CSS2B remains in the cell; 3) CSS1 and CSS2 variants may form a multiple complex; 4) whereas co-expression of CSS1 and CSS2A achieves CS chain polymerization in vitro, that of CSS1 and CSS2B does not; 5) by metabolic labeling analysis, whereas CSS2A facilitates CS biosynthesis, CSS2B inhibits it. Taken together, these results postulate a regulatory mechanism of CS biosynthesis by two CSS2 variants with different functions.
      As CSS2B is translated from the ATG-2 in the exon 2 of CSS2A, the β3-glycosyltransferase, β4-glycosyltransferase, and DXD motifs, critical for the catalytic function of CSS2A, are conserved in CSS2B. Although CSS2B exhibits the dual enzymatic activities of GalNAcT-II and GlcAT-II in vitro, its GlcAT activity is ∼26% that of CSS2A. Homology modeling of the N-terminal domain of both variants strongly suggests that removal of the region, as observed in CSS2B, abrogates a putative UDP-sugar binding site in N-CSS2A model and generates another putative UDP-sugar binding site. Therefore, the decrease of GlcAT activity in CSS2B is likely due to lack of an N-terminal region of 162 amino acid residues. Our in vitro assay has revealed that, whereas CSS2A co-expressed with CSS1 achieves CS polymerization, CSS2B with CCS1 does not. This suggests that CS polymerization requires a certain level of GlcAT activity of CSS2, or alternatively, the structure of the complex including CSS1 and CSS2B is different from the functional complex including CSS1 and CSS2A.
      It has been reported that any two of the CS glycosyltransferases (CSS1, CSS2, and CSS3) co-expressed in a cell form a complex and achieve polymerization; however, these enzymes, when individually expressed, do not exhibit polymerizing activity, suggesting that the formation of the functional enzyme complex requires any two of these enzymes in the cell (
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ). In this study a series of pulldown assays using co-expressed enzymes have revealed that any one of these enzymes pulled down another enzyme, consistent with the previous report (
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ). Moreover, we have observed that any of the CSS1 and CSS2 variants pulled down the same enzyme, implicating that these enzymes form the homogenous multimer. Our results suggest that individual enzymes do not have the specificity of partnership. As co-expression of two different enzymes is required for polymerization, there appears to be a mechanism by which different enzymes form a functional complex for efficient CS polymerization. Although our data do not eliminate the possibility that two different glycosyltransferases form a heterodimer rather non-specifically, they suggest that a functional complex for CS polymerization involves three or more molecules. Our results obtained by size exclusion chromatography support this, demonstrating CSS2 variants were eluted in fractions at substantially higher molecular mass (supplemental Fig. S2). Further studies remain to be performed to identify the other molecules that participate in formation of the functional complex of in vivo CS chain polymerization.
      Although CSS2B lacks the N-terminal transmembrane, its major proportion was localized in the cell, which is probably due to its interaction with other glycosyltransferases with an N-terminal transmembrane domain. Our immunocytochemical analysis has demonstrated that both CSS2 variants are localized in both the ER and the Golgi apparatus, which is consistent with previous reports (
      • Izumikawa T.
      • Koike T.
      • Shiozawa S.
      • Sugahara K.
      • Tamura J.
      • Kitagawa H.
      ). The presence of these variants in the ER is likely due to their overexpression in this system.
      CS is polymerized in chain length varying from 20 to 50 kDa, depending on the core protein, tissue location, age, and disease conditions (
      • Maeda N.
      • Fukazawa N.
      • Hata T.
      ,
      • Brown M.P.
      • Trumble T.N.
      • Plaas A.H.
      • Sandy J.D.
      • Romano M.
      • Hernandez J.
      • Merritt K.A.
      ,
      • Roughley P.J.
      • White R.J.
      ,
      • Mitchell D.
      • Hardingham T.
      ). Comparison of the CS chain length by different pairs of CSSs (
      • Izumikawa T.
      • Uyama T.
      • Okuura Y.
      • Sugahara K.
      • Kitagawa H.
      ) suggested that the length is dependent on the pair of CSSs co-expressed and that the pair of CSS1 and CSS2 polymerizes CS chains most efficiently. Among CSSs, CSS3 is expressed at quite low levels compared with CSS1 and CSS2 (
      • Yada T.
      • Gotoh M.
      • Sato T.
      • Shionyu M.
      • Go M.
      • Kaseyama H.
      • Iwasaki H.
      • Kikuchi N.
      • Kwon Y.D.
      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Narimatsu H.
      • Kimata K.
      ,
      • Yada T.
      • Sato T.
      • Kaseyama H.
      • Gotoh M.
      • Iwasaki H.
      • Kikuchi N.
      • Kwon Y.D.
      • Togayachi A.
      • Kudo T.
      • Watanabe H.
      • Narimatsu H.
      • Kimata K.
      ). Thus, it is likely that the pair of CSS1 and CSS2 plays the major role in CS chain polymerization. Our analysis of CS biosynthesis has clearly demonstrated substantial involvement of CSS2 in CS biosynthesis and has further demonstrated a distinct function of its variant, CSS2B. Overexpression of CSS2A facilitated CS biosynthesis, including chain polymerization in COS-7 cells (Fig. 6), indicating that the level of endogenous CSS2 is unsaturated in COS-7 cells and that overexpression of CSS2A increases the number of the functional complex of CS polymerization. In contrast, overexpression of CSS2B substantially inhibited endogenous CS biosynthesis, suggesting that CSS2B disturbs formation of the functional complex by competing with CSS2A. The opposite function of CSS2 variants suggests that CSS2 regulates CS chain polymerization by balancing the ratio of the variants.
      Molecular modeling predicted that CSS2B is structurally stable but CSS2B lacks an N-glycosylation site and a cysteine. Actually, we have detected not only the original CSS2A but also CSS2B variant as endogenous proteins. To date, chondroitin-4-sulfotransferase 1 (
      • Klüppel M.
      • Wight T.N.
      • Chan C.
      • Hinek A.
      • Wrana J.L.
      ) and a disease condition like arthritis (
      • Bollet A.J.
      • Nance J.L.
      ) are reported to affect the CS chain length as well as CS glycosyltransferases. Our observation that CS chains in brain become shorter during growth, concomitant with an increase in the ratio of CSS2B expression, suggests the biological relevance of CSS2B and presents another member participating in the regulation of CS chain length. Interestingly, the ratio of CSS2 variants expressed is different among tissues. Whereas MEFs express more than 95% of CSS2A, brain at 9 weeks expresses ∼75 and 25% of CSS2A and CSS2B, respectively. As CS chain length is known to vary among tissues, the ratio of CSS2 variants may be involved in the regulation of the length.
      It has not been determined whether CSS2 variants are present in other species, including humans. So far, no human cDNA comparable with mouse CSS2B has been identified. As shown in Fig. 1, CSS2B has an additional exon 1′ in intron 1. The human genomic locus of CSS2 contains a putative exon corresponding to mouse exon 1′, exhibiting 76% nucleotide sequence identity. This region has a consensus GT-AG splice sequence and some exonic splicing enhancer sequences. These data strongly suggest the presence of exon 1′ in humans. The putative exon 1′ of the human CSS2 genome contains ATG, and if translation initiates at this ATG, a longer variant with a N-terminal region of 55 amino acid residues different from the putative human CSS2B would be synthesized. Although the human database has no information showing transcription of the putative exon 1′, the Ensembl database (
      • Hubbard T.J.
      • Aken B.L.
      • Ayling S.
      • Ballester B.
      • Beal K.
      • Bragin E.
      • Brent S.
      • Chen Y.
      • Clapham P.
      • Clarke L.
      • Coates G.
      • Fairley S.
      • Fitzgerald S.
      • Fernandez-Banet J.
      • Gordon L.
      • Graf S.
      • Haider S.
      • Hammond M.
      • Holland R.
      • Howe K.
      • Jenkinson A.
      • Johnson N.
      • Kahari A.
      • Keefe D.
      • Keenan S.
      • Kinsella R.
      • Kokocinski F.
      • Kulesha E.
      • Lawson D.
      • Longden I.
      • Megy K.
      • Meidl P.
      • Overduin B.
      • Parker A.
      • Pritchard B.
      • Rios D.
      • Schuster M.
      • Slater G.
      • Smedley D.
      • Spooner W.
      • Spudich G.
      • Trevanion S.
      • Vilella A.
      • Vogel J.
      • White S.
      • Wilder S.
      • Zadissa A.
      • Birney E.
      • Cunningham F.
      • Curwen V.
      • Durbin R.
      • Fernandez-Suarez X.M.
      • Herrero J.
      • Kasprzyk A.
      • Proctor G.
      • Smith J.
      • Searle S.
      • Flicek P.
      ) shows that the mRNA sequence that encodes CSS2A exhibits another translation initiation site that may synthesize CSS2B in mouse (Ensembl gene accession no. OTTMUS600000019412). Similarly, the mRNA of human CSS2 may be used for the synthesis of CSS2B as well as CSS2A.
      Our study proposes a mechanism of regulation of CS biosynthesis by CSS2 variants. Characterization of CS glycosyltransferases should involve their splice variants because they may exhibit distinct functions. Systematic analysis using recombinant enzymes, deduced by cDNAs showing putative variants, will lead to determination of the enzyme complex and elucidation of the mechanisms of CS biosynthesis. In addition, our findings implicate that CSS2B may serve as a useful tool for regulation of CS biosynthesis. As inhibition of expression of a glycosyltransferase may not suppress CS biosynthesis due to compensation by other CSSs, overexpression of CSS2B may be applied as an alternative method to suppression of CS biosynthesis.

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