Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

Abstract

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant. The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant. Heparan sulfates (HSs) 4The abbreviations used are: HS, heparan sulfate; EXT, exostosin; GAG, glycosaminoglycan; GlcA, d-glucuronic acid; HEK, human embryonic kidney; HME, Hereditary Multiple Exostoses; NDST, N-deacetylase/N-sulfotransferase; siRNA, small interfering RNA; TI, transferase I; TII, transferase II; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography. are sulfated glycosaminoglycans (GAGs) distributed on the cell surfaces and in the extracellular matrices of most tissues. HS chains are synthesized covalently linked to various proteins in a proteoglycan structure. HS proteoglycans have been implicated as regulators in a diverse number of biological events related to intracellular signaling, cell-cell interactions, and tissue morphogenesis (1Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (399) Google Scholar). Most of these activities depend on the binding of the negatively charged HS chains to a variety of molecules including members of the fibroblast growth factor family, serine protease inhibitors, and extracellular matrix proteins (2Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 3Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1243) Google Scholar). The biosynthesis of HS is a complex process (reviewed in Refs. 2Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 3Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1243) Google Scholar, 4Esko J.D. Lindahl U. J. Clin. Invest. 2001; 108: 169-173Crossref PubMed Scopus (788) Google Scholar). Chain elongation is initiated by the formation of a tetrasaccharide linkage region composed of glucuronic acid-galactose-galactose-xylose (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-), where Xyl is attached to a serine residue in the core protein (5Sugahara K. Kitagawa H. IUBMB Life. 2002; 54: 163-175Crossref PubMed Scopus (217) Google Scholar). After the addition of a single GlcNAc residue by GlcNAc transferase I (GlcNAc-TI), elongation proceeds by the action of glycosyltransferases (GlcNAc-TII and GlcA-TII), which add β1,4-GlcA and α1,4-GlcNAc units in alternating sequence to the nonreducing end of the growing polymer (6Lidholt K. Lindahl U. Biochem. J. 1992; 287: 21-29Crossref PubMed Scopus (60) Google Scholar). The polymerization reactions are carried out by the exostosin proteins EXT1 and EXT2 (7McCormick C. Duncan G. Goutsos K.T. Tufaro F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 668-673Crossref PubMed Scopus (372) Google Scholar, 8Senay C. Lind T. Muguruma K. Tone Y. Kitagawa H. Sugahara K. Lidholt K. Lindahl U. Kusche-Gullberg M. EMBO Rep. 2000; 1: 282-286Crossref PubMed Scopus (143) Google Scholar, 9Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (147) Google Scholar). Concomitantly with chain elongation modification of the chain occurs, initiated by N-deacetylation and N-sulfation of glucosamine units, carried out by the bifunctional enzyme N-deacetylase/N-sulfotransferase (NDST). These reactions occur in a more or less blockwise fashion and create stretches of N-sulfated regions and nonmodified N-acetylated domains, interspersed with short mixed regions (N-sulfated/N-acetylated domains) (10Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar, 11Maccarana M. Sakura Y. Twada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The N-deacetylation/N-sulfation reaction has been regarded as a key regulatory step because all subsequent modifications, epimerization of GlcA to iduronic acid and various sulfation reactions, only occur in the vicinity of N-sulfate groups. However, NDST1-/-/NDST2-/- embryonic stem cells that completely lack N-sulfation are still able to produce 6-O-sulfated HS (12Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellen L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Subsequent modifications include C-5 epimerization of GlcA to iduronic acid, 2-O-sulfation of iduronic acid, and 6-O-sulfation of N-acetylated and N-sulfated GlcN residues. In addition, a small number of N-sulfated d-glucosamine residues become O-sulfated at C-3, and a few GlcA units become O-sulfated at C-2. Five members of the EXT family of glycosyltransferases are known in humans: EXT1, EXT2, EXTL1 (for EXT-like 1), EXTL2, and EXTL3. Members of the EXT gene family encode glycosyltransferases that are suggested to be involved in HS biosynthesis, and most of the members seem to possess more than one in vitro glycosyl transferase activity (5Sugahara K. Kitagawa H. IUBMB Life. 2002; 54: 163-175Crossref PubMed Scopus (217) Google Scholar, 9Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (147) Google Scholar). EXT1 and EXT2 appear to have dual enzyme activities, GlcA-TII and GlcNAc-TII, although those of EXT2 are weak and have been questioned. Even though EXT1 alone is able to polymerize the HS backbone structure in vitro, all evidence suggests that EXT1 and EXT2 are chain elongation M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Kitagawa H. J. J. Sugahara K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). catalyze GlcNAc transferase EXTL1 GlcNAc to the nonreducing end of the growing (GlcNAc-TII EXTL3 GlcNAc to the protein linkage region and to the growing chain (GlcNAc-TII enzyme activities involved in the and elongation of HS chains Kitagawa H. J. T. Kusche-Gullberg M. Lindahl U. Sugahara K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google and EXTL2, the of the EXT family, activity H. H. Sugahara K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The biological roles of EXTL1 and in HS biosynthesis have not been and in to the members of the EXT family, of the EXTL1 and are in that they are not HS biosynthesis. Hereditary multiple hereditary is one of the most with a The are shorter and by bony outgrowths at the epiphyseal end of the L. J. Google Scholar). The by which is in either EXT1 or EXT2 and the or of HS in the has been implicated in in J.T. M. E. 2002; PubMed Scopus Google Scholar, J.T. E. PubMed Scopus Google Scholar). number of in EXT1 and EXT2 have been in and the of in EXT1 of the B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (147) Google Scholar, C. A. M. A. J. L. J. 2001; PubMed Scopus Google Scholar). The are distributed the EXT1 whereas EXT2 appear to be the of the protein. Most in of and of are single The acid are believed to the of the EXT proteins. However, although it is believed that the in HS this is not the of the EXT1 mutant proteins to HS C. Crawford B.E. Esko J.D. Tufaro F. Duncan G. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the human embryonic kidney 293 we the of EXT1, EXT2, and EXTL3 to HS chain elongation by of by overexpression and of by small interfering RNAs We also the effect on HS of EXT2 with in with human EXT1 and EXT2 and EXTL3 and and siRNA, were 293 cells were transfected with the of to the After the was and the cells were at in with or were as was cells transfected with or control siRNA was generated by of was and The were PubMed Scopus Google Scholar). The used EXT1 and EXT2 and and EXTL3 and were against of and was in with was the 2001; PubMed Scopus Google Scholar). of and of 293 to the EXT1 or EXT2 was a the and and and The was cloned into and The EXT1 and EXT2 were either or into the were introduced into EXT2 the to the was and The EXT2-Y419X mutant was generated and are and in The wild type and were with and into the of the with the region in to were by form of human the was a human with the and and the were cloned with the sequence and the The truncated of EXT1 and EXT2 were as M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). type and mutant were or transfected into cells the EXT1, EXT2, and and control transfected with alone or were as J. H. K. Lindahl U. Kusche-Gullberg M. Biochem. J. 2000; PubMed Google Scholar). were in with and or at of and and of HS Cell transfected cell were with or the in the After at the was and the cells were with phosphate-buffered and with at and the cell were The were and with at to the the protein The were to in The were with of the or The were with and were with in and and the reaction were by on a with HS was with I and in and To HS, the cells were in and protease and of at After at the the proteoglycans was and by chromatography. After on chains were the and with and The were with and as of HS chains, to a of was to the After at the was and to a of chains were on as of HS chains were with acid at by with PubMed Scopus Google Scholar). The were by on in to and were and by were by a with of at a of E. H. E. H. K. Lindahl U. Kusche-Gullberg M. Biochem. J. PubMed Google Scholar). and GlcA transferase activities were as M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activities of cell were by of protein with of and of is to After at the were to of in were by activities were in that was used as a and were used as 293 cells were transfected with on as the cells were with PBS, with in with PBS, in in and with in The cells were with the and the by of at of and at were in and further with the and they were in with The were a with and a of EXT1, EXT2, and investigate the role of EXT1, EXT2, and EXTL3 in HS chain we transfected 293 cells with siRNA EXT1 EXT2 EXTL3 and by the EXT were to of that of the control cells. The cells transfected with either or GlcNAc and GlcA transferase activities to the control cells. of cells with EXTL3 siRNA increased transferase activities We were to was a of EXT1, EXT2, and EXTL3 at the we also of EXT1, EXT2, or EXTL3 the of the by no detectable effect of of one EXT on the of the not of of EXT1, EXT2, and EXTL3 on HS Chain investigate the effect of siRNA on HS 293 cells with or the control siRNA were with or were the cell and the and the were the core proteins. The cells transfected with EXTL3 siRNA increased chain length, and those transfected with EXT1 and EXT2 siRNA HS chains that were shorter than those synthesized by the control cells and cells with a sulfate to the control transfected whereas the in cells was Interestingly, the effect of EXT1 or EXT2 on HS chain elongation is to HS by embryonic a in EXT1 so that they still small of EXT1 S. M. M. Sugahara K. Kusche-Gullberg M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The mutant cells produce HS with wild type and the HS chain length, although in the number of chains also have S. M. M. Sugahara K. Kusche-Gullberg M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). of of EXT1, EXT2, and EXTL3 on HS we with more than in by and with a effect on HS chain The various were with to in the were and the of HS cells were by of with acid at that in of the chains at the of N-sulfated glucosamine these N-acetylated units N-sulfated be to whereas alternating N-sulfated and N-acetylated glucosamine residues to with N-sulfate of at The of the of the sulfated regions with a of However, it was that the of EXT1 and EXT2 resulted in more pronounced of and than HS control cells. the of was by of the generated by of HS The were by into O-sulfated was a small 6-O-sulfated in and HS with the control the N-sulfated of HS and transfected cells HS were to at by of with that were by of the of N-sulfated d-glucosamine iduronic the of N-sulfated d-glucosamine iduronic acid in a of Overexpression of EXT1, EXT2, and EXTL3 on HS Chain the effect of overexpression of EXT1 and EXT2 on HS chain 293 cells were transfected with the alone EXT1, EXT2, or EXT1 and EXT2 The GlcNAc and GlcA transferase activities of the EXT1 overexpressing cells on a protein were increased with the activities transfected cells and to C. Lind T. Muguruma K. Tone Y. Kitagawa H. Sugahara K. Lidholt K. Lindahl U. Kusche-Gullberg M. EMBO Rep. 2000; 1: 282-286Crossref PubMed Scopus (143) Google of EXT1 and EXT2 further the EXT1, EXT2, and overexpressing cells were and HS proteoglycans were by on and the HS chains were the core protein by and L. Scholar). in EXT1 overexpression resulted in a in chain length, which was even more in cells. HS EXT2 or EXTL3 overexpressing cells no in chain length control even though in chain length were The effect on chain elongation was not by the because the effect on HS chain elongation was also cells were or the chains HS was by the of to and by with a of and I and not of overexpression of of the EXT proteins on HS chain proteoglycans were transfected 293 and the chains were and further as After with the were by on a are of cells overexpressing truncated EXT1, EXT2, or or EXT2, or EXT2-Y419X The in are at cell the of cells overexpressing EXT1, or The cells were with and In all of the the are with transfected control cells overexpressing The to that is by The EXT proteins are type and to the on HS chain elongation were on the of the we the effect on HS chain length of overexpression of EXT1, EXT2, and EXTL3. 293 cells were transfected with human EXT1, and EXT2, or with of the the domains, proteins that were into the The proteins were on and the proteins were The activities of EXT1, EXT2, and have been M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The EXTL3 weak activity and no detectable GlcA-TII activity not The weak activity be by the although EXTL3 has been to GlcNAc to activity the HS protein linkage region is Kitagawa H. J. T. Kusche-Gullberg M. Lindahl U. Sugahara K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). Overexpression of EXT1 and resulted in a in chain length, whereas overexpression of EXT2 or EXTL3 not HS chain elongation However, in to the with the of EXT2 and EXT1 not enhance HS chain length more than EXT1 These that the activities of EXT1 not depend on the region of the EXT1 protein. of transfected cells that the proteins were also in the cell EXT1 protein is able to with the in the of Overexpression of EXT on HS Chain the effect of EXT2 in this we generated 293 cell transfected with and EXT2-Y419X have been to as of number of exostoses, and C. A. M. A. J. L. J. 2001; PubMed Scopus Google Scholar). One of the that we is to a The EXT2-Y419X, a in a truncated a of transfected cells were GlcA and GlcNAc transferase The enzyme activities of the cell lines the EXT2 were to those of transfected cells not that overexpression of or wild type EXT2 not the in vitro high of EXT2, and EXT2-Y419X were by and used to investigate the effect of EXT2 on HS The 293 cell were with and were cells and the and of wild type or of EXT2 not the of the as with transfected cells not to the EXT2 overexpressing the cells overexpressing of EXT2 no in chain length with HS in transfected cells that the EXT2 proteins not exhibit a effect on HS We the EXT2 the capacity of EXT2 on HS chain elongation with 293 cells were transfected with EXT1 or EXT2-Y419X alone or with EXT1 and EXT2 or with EXT1 and EXT2-Y419X of HS cells transfected with EXT1 and that the most HS chains were in cells coexpressing EXT1 and EXT2 In not chain elongation more than EXT1 alone were of EXT2-Y419X into transfected EXT1 cell not of the transfected cells that of EXT2 and EXT2-Y419X, were in the transfected cells not the lack of effect of the EXT2 mutant was not to less EXT2 protein to the effect on chain of not the transferase activities and of the EXT to the this of HS chain elongation of 293 cells transfected with we the transfected cells with against EXT2 to the of the proteins. in EXT2 and EXT2-Y419X proteins with the the lack of effect of is not by of the protein in the of the of HS is is The of HS chain elongation is not and the role of EXT and EXTL protein is still of have been to possess in vitro activities HS chain or elongation (5Sugahara K. Kitagawa H. IUBMB Life. 2002; 54: 163-175Crossref PubMed Scopus (217) Google Scholar, 9Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (147) Google Scholar). to the of these proteins is to the of we the role of EXT1, EXT2, and EXTL3 in HS biosynthesis by of overexpression and gene The most were the of of the EXT proteins on HS chain elongation. We have that embryonic a in shorter HS chains than wild type S. M. M. Sugahara K. Kusche-Gullberg M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). with of EXT1 by siRNA in human cells resulted in activities and the formation of shorter HS chains Interestingly, of EXT2 also resulted in the of shorter chains, that EXT2 is a of EXT1 in chain elongation. To more into the role of EXT2 in HS we also the effect on HS chain elongation of cell lines overexpressing wild type and of EXT2 alone or with in either EXT1 or EXT2 all evidence suggests that EXT1 and EXT2 are HS it is believed that in HS the related to EXT1 and EXT2 are and in tissues. However, the of seem to be to the growing the high of the EXT2 they not HS that the EXT2 with to and EXT2-Y419X, a acid protein the most acid not exhibit a Interestingly, of mutant EXT1 into cells also not HS G. Esko J.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). these that the of EXT proteins is not by the of increased of mutant of EXT1 or EXT2. to the with the EXT2, of the wild type EXT2 with EXT1 in cells had a effect on enzyme activity and HS chain length and The effect of EXT2 on chain elongation is EXT2 is HS polymerization as in and of EXT2 Duncan Selleck S. 2004; PubMed Scopus Google Scholar, C. M. M. 2004; PubMed Scopus Google Scholar, Y. Y. M. A. T. 2004; PubMed Scopus Google Scholar, S. C. Selleck S.B. H. 2004; Full Text Full Text PDF PubMed Scopus (143) Google Scholar, D. B.M. Esko J.D. PubMed Scopus Google Scholar). in vitro that EXT1 has detectable GlcA and GlcNAc transferase activities, whereas the enzyme activities of EXT2 are less (7McCormick C. Duncan G. Goutsos K.T. Tufaro F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 668-673Crossref PubMed Scopus (372) Google Scholar, 8Senay C. Lind T. Muguruma K. Tone Y. Kitagawa H. Sugahara K. Lidholt K. Lindahl U. Kusche-Gullberg M. EMBO Rep. 2000; 1: 282-286Crossref PubMed Scopus (143) Google Scholar, M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The lack of effect of EXT2 overexpression be to the that EXT2 have a in HS polymerization EXT1 and EXT2 were one gene and overexpression to proteins. EXT2 a complex with EXT1, which the of proteins to the and the activity of the protein (7McCormick C. Duncan G. Goutsos K.T. Tufaro F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 668-673Crossref PubMed Scopus (372) Google Scholar, 8Senay C. Lind T. Muguruma K. Tone Y. Kitagawa H. Sugahara K. Lidholt K. Lindahl U. Kusche-Gullberg M. EMBO Rep. 2000; 1: 282-286Crossref PubMed Scopus (143) Google Scholar, M. Kusche-Gullberg M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the is believed to be the HS of the transferase activities of EXT2, it is to that EXT2 is not involved in the elongation of the HS backbone the of EXT2 be in the and of EXT1 to the One of be that of EXT2 protein is synthesized in the increased of EXT2 be effect and not the of EXT1 are more form and chain elongation. Overexpression of EXT1 and EXT2 even more that in chain elongation. is not known the complex of EXT1 and EXT2 is the mutant to the it is the mutant EXT2 to with EXT1 and was not able to further chain elongation. are to the role of EXT2 in HS we that chain length than the of HS chains is by the of EXT1 and Interestingly, NDST1-/-/NDST2-/- embryonic stem which lack increased HS chain length the of HS by the a more complex of chain elongation (12Holmborn K. Ledin J. Smeds E. Eriksson I. Kusche-Gullberg M. Kjellen L. J. Biol. Chem. 2004; 279: 42355-42358Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In to the of EXT1 or EXT2, of EXTL3 resulted in the of longer HS chains. protein has been to be involved in HS chain the of the GlcNAc the protein linkage region Kitagawa H. J. T. Kusche-Gullberg M. Lindahl U. Sugahara K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). The in HS chain length EXTL3 be by the that the in EXTL3 in linkage regions that the GlcNAc to the HS elongation process where the GlcNAc is in less more polymerization occur in the chains that are The lack of effect of EXTL3 overexpression be to the that the cells produce of which is to catalyze the of GlcNAc residues in all of the linkage has also been that EXTL3 be involved in chain elongation or chain Kitagawa H. J. T. Kusche-Gullberg M. Lindahl U. Sugahara K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). the of a chain to longer HS chains, overexpression of EXTL3 not the HS chain length as have been EXTL3 was a of HS chains. In addition, cells activity still activity lack HS chains G. Esko J.D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google this mutant a with the structure L. Esko J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the that EXTL3 catalyzes the than the of HS chain elongation. has been in a that a GlcA residue is at the nonreducing end of the HS chains M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). EXTL3 no activities Kitagawa H. J. T. Kusche-Gullberg M. Lindahl U. Sugahara K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google so it be to catalyze chain In that siRNA of EXT1, EXT2, or EXTL3 HS chain which be one the formation of exostoses, because the shorter chains by the EXT1 growth factor these proteins and roles in HS biosynthesis in human cells. EXT1 and EXT2 are together chain and the of the proteins the polymerization The of EXTL3 also chain the that or EXTL3 be of HS chains. We