A Soluble Form of α1,3-Galactosyltransferase Functions within Cells to Galactosylate Glycoproteins

Abstract

It has been assumed that membrane-bound glycosyltransferases function within the Golgi apparatus to glycosylate glycoproteins. We now report, however, that a truncated, soluble recombinant form of the murine α1,3-galactosyltransferase expressed in human 293 cells is highly efficient and comparable to the full-length enzyme in α-galactosylating both newly synthesized membrane-associated and secreted glycoproteins. Although the soluble enzyme was secreted by cells as expected, we also found that the full-length, membrane-associated form was secreted. Unexpectedly, both secreted forms are cleaved identically at two primary sites within the stem region by endogenous protease(s) at the indicated positions in the sequence 73KDWW↓FPS↓WFKNG. These results demonstrate that the soluble α1,3-galactosyltransferase is functional within the cell and that specific proteolysis occurs in the stem region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal glycoconjugate biosynthesis may involve cooperation between membrane-bound and soluble enzymes. It has been assumed that membrane-bound glycosyltransferases function within the Golgi apparatus to glycosylate glycoproteins. We now report, however, that a truncated, soluble recombinant form of the murine α1,3-galactosyltransferase expressed in human 293 cells is highly efficient and comparable to the full-length enzyme in α-galactosylating both newly synthesized membrane-associated and secreted glycoproteins. Although the soluble enzyme was secreted by cells as expected, we also found that the full-length, membrane-associated form was secreted. Unexpectedly, both secreted forms are cleaved identically at two primary sites within the stem region by endogenous protease(s) at the indicated positions in the sequence 73KDWW↓FPS↓WFKNG. These results demonstrate that the soluble α1,3-galactosyltransferase is functional within the cell and that specific proteolysis occurs in the stem region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal glycoconjugate biosynthesis may involve cooperation between membrane-bound and soluble enzymes. The synthesis of the carbohydrate groups in secreted and membrane-bound glycoconjugates occurs in the endoplasmic reticulum and Golgi apparatus and involves glycosyltransferases, which utilize sugar nucleotides imported into the lumen of these organelles by specific import proteins (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Google Scholar, 2Hirschberg C.B. Snider M.D. Annu. Rev. Biochem. 1987; 56: 63-88Google Scholar, 3Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Google Scholar, 4Kleene R. Berger E.G. Biochim. Biophys. Acta. 1993; 1154: 283-325Google Scholar). Most of the cloned mammalian glycosyltransferases share many features in common, although their primary structures are often unrelated. The deduced amino acid sequences of the cDNA clones encoding many glycosyltransferases reveal that they are type 2 transmembrane proteins containing a short N-terminal cytoplasmic tail consisting of 4–24 amino acids, a membrane-spanning domain, and an extended stem region in the lumen that precedes the large C-terminal catalytic domain (4Kleene R. Berger E.G. Biochim. Biophys. Acta. 1993; 1154: 283-325Google Scholar, 5Field M.C. Wainwright L.J. Glycobiology. 1995; 5: 463-472Google Scholar). The transmembrane domain and flanking sequences of many glycosyltransferases may be important in retaining/targeting the enzymes in the Golgi apparatus (6Teasdale R.D. D'Agostaro G. Gleeson P.A. J. Biol. Chem. 1992; 267: 4084-4096Google Scholar, 7Russo R.N. Shaper N.L. Taatjes D.J. Shaper J.H. J. Biol. Chem. 1992; 267: 9241-9247Google Scholar, 8Nilsson T. Lucoq J.M. Macay D. Warren G. EMBO J. 1991; 10: 3567-3575Google Scholar, 9Aoki D. Lee N. Yamaguchi N. Dubois C. Fukuda M.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4319-4323Google Scholar, 10Tang B.L. Wong S.H. Lowe S.H. Hong W. J. Biol. Chem. 1992; 267: 10122-10126Google Scholar, 11Burke J. Pettitt J.M. Humphris D. Gleeson P.A. J. Biol. Chem. 1994; 269: 12049-12059Google Scholar, 12Munro S. EMBO J. 1991; 10: 3577-3588Google Scholar, 13Colley K.J. Lee E.U. Paulson J.C. J Biol. Chem. 1992; 267: 7784-7793Google Scholar, 14Wong S.H. Low S.H. Hong W. J. Cell. Biol. 1992; 117: 245-258Google Scholar, 15Masibay A.S. Balaji P.V. Boeggeman E.E. Qasba P.K. J. Biol. Chem. 1993; 268: 9908-9916Google Scholar, 16Dahdal R.Y. Colley K.J. J. Biol. Chem. 1993; 268: 26310-26319Google Scholar, 17Munro S. EMBO J. 1995; 14: 4695-4705Google Scholar). However, the exact mechanisms and the precise purpose of the targeting have not been elucidated. Because of their transmembrane nature, it has been presumed that Golgi glycosyltransferases function in the membrane-anchored form. Numerous glycosyltransferases have been detected, however, as soluble forms in serum, milk, colostrum, and/or growth media from normal and transformed cell lines (18Berger E.G. Kozdrowski I. Weiser M.M. van den Eijnden D.H. Schiphorst W.E. Eur. J. Biochem. 1978; 90: 213-222Google Scholar, 19Hudgin R.L. Schachter H. Can. J. Biochem. 1971; 49: 829-837Google Scholar, 20Bartholomew B.A. Jourdian G.W. Roseman S. J. Biol. Chem. 1973; 248: 5751-5762Google Scholar, 21Klohs W.D. Mastrangelo R. Weiser M.M. Cancer Res. 1981; 41: 2611-2615Google Scholar, 22Serafini-Cessi F. Malagolini N. Guerrini S. Turrini I. Glycoconj. J. 1995; 12: 773-779Google Scholar, 23Debose-Boyd R.A. Nyame A.K. Smith D.F. Cummings R.D. Arch. Biochem. Biophys. 1996; 335: 109-117Google Scholar). The mechanism(s) of formation and secretion of soluble and active glycosyltransferases are not understood. N-terminal sequence analyses of soluble forms of the α2,6-sialyltransferase and β1,4-galactosyltransferase demonstrate that they arise through proteolysis within the stem region, thereby releasing the catalytic domain from the membrane (24D'Agostaro G. Bendiak B. Tropak M. Eur. J. Biochem. 1989; 183: 211-217Google Scholar, 25Weinstein J. Lee E.U. McEntee K. Lai P.-H. Paulson J.C. J. Biol. Chem. 1987; 262: 17735-17743Google Scholar). The proteases responsible for cleaving the enzymes are not defined, although evidence suggests that a cathepsin D-like protease may be responsible for the cleavage of α2,6-sialyltransferase (26Lammers G. Jamieson J.C. Biochem. J. 1989; 261: 389-393Google Scholar). The functions of the soluble glycosyltransferases are not known. One enzyme that has recently been shown to be secreted from cultured cells is the murine α1,3-galactosyltransferase (α1,3GT) 1The abbreviations used are: α1,3GT, UDP-Gal:β-d-Gal α1,3-galactosyltransferase; HA, influenza hemagglutinin; TBS, Tris-buffered saline; GS-I-B4, Griffonia simplicifoliaI-B4 isolectin; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar, 28Cho, S. K., Yeh, J.-C., and Cummings, R. D. (1997) Glycoconj. J. in press.Google Scholar), which is responsible for the synthesis of terminal α-Gal sequences in Galα1 → 3Galβ1 → 4GlcNAc-R. The murine (29Larsen R.D. Rajan V.P. Ruff M.M. Kukowska-Latallo J. Cummings R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8227-8231Google Scholar), bovine (30Joziasse D.H. Shaper J.H. Van den Eijnden D.H. Van Tunen A.J. Shaper N.L. J. Biol. Chem. 1989; 264: 14290-14297Google Scholar), porcine (31Sandrin M.S. Dabkowski P.L. Henning M.M. Mouthouris E. McKenzie I.F.C. Xenotransplantation. 1995; 11: 81-88Google Scholar, 32Strahan K.M. Gu F. Preece A.F. Gustavsson I. Andersson L. Gustafsson K. Immunogenetics. 1995; 41: 101-105Google Scholar), and New World monkey (33Henion T.R. Macher B.A. Anaraki F. Galili U. Glycobiology. 1994; 4: 192-201Google Scholar) cDNA encoding for α1,3GT have been identified. The α1,3GT is expressed in a variety of mammalian species, but it is not expressed in Old World monkeys, apes, and humans (34Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1984; 259: 9858-9866Google Scholar, 35Galili U. Shohet S.B. Korbin E. Stults C.L.M. Macher B.A. J. Biol. Chem. 1988; 263: 17755-17762Google Scholar). The expression of the α1,3GT in murine F9 teratocarcinoma cells is transcriptionally up-regulated by retinoic acid induction, but most of the newly synthesized enzyme is eventually secreted into the culture media of the cells (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar). The α1,3GT is secreted by many cell types, since we have detected soluble forms of the α1,3GT in the culture media of all cell lines expressing the and we have found enzyme in the of different expressing the functional α1,3GT S. K., Yeh, J.-C., and Cummings, R. D. (1997) Glycoconj. J. in press.Google Scholar). The occurrence of the α1,3GT in a soluble form to it may have a One is that the soluble enzyme may have a within the Golgi we that a truncated, soluble recombinant form of α1,3GT expressed in human 293 cells is highly efficient in α-galactosylating both newly synthesized membrane-associated and secreted and that it is comparable to the enzyme in both the and full-length enzymes are secreted from 293 cells and both are cleaved by endogenous protease(s) at two primary sites in the stem region. These results demonstrate that a soluble function within cells and that normal proteolysis of membrane-bound glycosyltransferases may be important in enzymes that in glycoconjugate Griffonia and fluorescein from was from enzymes from was from The was from was from The was from culture from used of the teratocarcinoma F9 cells cultured in containing culture as S. Cell. 1978; Scholar). of cells in media acid for 293 cells cultured in containing expressing the large cultured in media containing serum, and cells a of B. Lowe of C-terminal of the murine α1,3GT to a acid from influenza was by the and the as A. S. A. R. D. Cummings, and K. W. in the which the cDNA of the murine α1,3GT (29Larsen R.D. Rajan V.P. Ruff M.M. Kukowska-Latallo J. Cummings R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8227-8231Google Scholar), was used as the for the The containing a at the and a at the was into the and α1,3GT cDNA in was and and the was the encoding the The acid is by the P.A. R.A. 1991; Scholar). full-length was into 293 the from in a to the by the cell lines from the analyses (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar), and a that cell glycoconjugates was and form of the full-length α1,3GT was in which the stem sequence was to at sites for cleavage was by the and the the was to amino acid and to The which the full-length was used as the for the The was and and into of by into the was to the The containing the was into 293 cells the was in the of as was and culture media of as and a that expressed the α1,3GT was and a soluble form of murine a containing a for the and the catalytic domain of the enzyme was in the mammalian expression J. Biol. Chem. 1992; 267: Scholar). the domain of the α1,3GT was by the a and the a The which the cDNA of the murine α1,3GT S. K., Yeh, J.-C., and Cummings, R. D. (1997) Glycoconj. J. in press.Google Scholar), was used as the for the The was and and into of in a of the soluble α1,3GT to the sequence in the was into human 293 cells and was in the of as (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar), and lines that secreted enzyme into media and and The was in the mammalian expression J. Biol. Chem. 1992; 267: Scholar). both the N-terminal cytoplasmic and the transmembrane domain of and the domain of the was by the and the The cDNA encoding the murine used as the for the was a of and of The was and and into of in a of the soluble to the sequence in the was into human 293 cells and was in the of as was culture media of and lines that secreted into media in the of the protease and and as (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar, R.D. J. Biol. Chem. 1988; 263: Scholar). cell culture media for enzyme and cells in media they and to The media of culture and to for and the was at in a was from and was used at a of was a from and was used at a of α1,3GT the murine α1,3GT, a was that the domain of the murine α1,3GT and a at the the domain of the α1,3GT, amino acid was by the and the a The which the cDNA of the murine α1,3GT S. K., Yeh, J.-C., and Cummings, R. D. (1997) Glycoconj. J. in press.Google Scholar), was used as the for the The was and and into the of in a of The containing at the and a at the was into the and was used to into and the expressed was a acid to the by the The α1,3GT was by an the of the and used at a of The and used at of The secreted form of α1,3GT was from culture media of human 293 by a of J. Biol. Chem. 261: Scholar). media from and cells the cells and at of The was to a containing and A. was by containing the of at The enzyme was in and enzyme was in The enzyme was Scholar) and membrane The to the α1,3GT was the by and and to N-terminal amino sequence by the of and at in of for and by cell as (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar). The α1,3GT was by from culture media as was by The proteins in The proteins H. T. J. Proc. Natl. Acad. Sci. U. S. A. Scholar) and by The are as containing at for and they primary in containing bovine for in the at for in in by in and the to the the of α1,3GT from the and soluble in the culture media in and by the of of the and of soluble proteins in culture media as (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar) and to The proteins and by The was as (27Cho S.K Yeh J. Cho M. Cummings R.D. J. Biol. Chem. 1996; 271: 3238-3246Google Scholar) that the detected the of the soluble form of murine α1,3GT and to it to the full-length proteins as and in full-length α1,3GT was in which the acid influenza P.A. R.A. 1991; Scholar) was to the C-terminal of full-length is shown in and is the soluble form of the α1,3GT was in which the of the an J. Biol. Chem. 1992; 267: Scholar) and by amino of the murine α1,3GT encoding the C-terminal catalytic is shown in and is the 293 cells the and expressing 293 cell lines as One expressing was clones expressing the and these and expression of terminal Galα1 → expression of the α1,3GT (29Larsen R.D. Rajan V.P. Ruff M.M. Kukowska-Latallo J. Cummings R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8227-8231Google Scholar). The expressing cell lines for their to cell of enzyme and is a that terminal Galα1 → S. J. Biol. Chem. 1981; Scholar). expected, the 293 cell not Galα1 → 293 cells The of expression of Galα1 → in the expressing clones comparable to the normal in retinoic F9 cells cells These demonstrate that the soluble α1,3GT in biosynthesis of glycoconjugates containing the Galα1 → these analyses and the of GS-I-B4, the soluble α1,3GT to be comparable to the full-length enzyme in Galα1 → are and/or between the full-length soluble forms of the enzyme in the they glycosylate the and media from the clones and expected, the from 293 cell and media not of membrane-associated was in of and although the of expression in However, in the of membrane-associated in between the clones expressing full-length enzyme both the and the of in the media of all the clones all the of to was since of sugar and as expected, to a of which is to terminal R.G. M. R.A. Biochim. Biophys. Acta. Scholar, R.N. F. 1989; Scholar). These results demonstrate that both the full-length and soluble α1,3GT the biosynthesis of of glycoproteins. are in the of expression of the the full-length α1,3GT and are in secretion of the two we the enzyme in cell and culture media as the and as the The results are shown in was detected in of the 293 α1,3GT was in of cells but of the which in the cell culture was in the culture media The of the α1,3GT in of and cells that of cell be expected, the of of α1,3GT in media of and cells that in the media from in cell and media from human 293 cells full-length of the α1,3GT was as the and as the as The of the α1,3GT was as the and as the as in a We the that the secreted to it was secreted from the from secreted as the 293 cells to and the media by culture media of The enzyme in the media was 293 cells media from 293 cells not expressing the of media containing α1,3GT the cells and for Galα1 → and was expression of Galα1 → in cells to the and between 293 which been to and the cells not These demonstrate that Galα1 → in cells expressing the α1,3GT arise from enzyme within the cells from of the by the secreted enzyme the that the full-length enzyme was in a soluble form into the the α1,3GT in cells was by the The α1,3GT in as a at in the of the α1,3GT from media is These demonstrate that the full-length α1,3GT is expressed by the cells and secreted as a soluble form and that both forms the at their We also to the soluble form of the α1,3GT expressed from cells and Unexpectedly, the secreted form of the enzyme from cells not the The recombinant was as shown by in a murine The secreted enzyme an These results that the recombinant α1,3GT the secretion of the We the that the was cleaved from the by a protease(s) that in the the recombinant recombinant was and expressed in 293 293 cells cDNA encoding a form of the in which the was to the in a as is a expressed murine cells R. M. J. 1991; Scholar). It is a type 2 transmembrane and in it glycosyltransferases in within the from cells expressing These results demonstrate that the is recombinant that the is cleaved from the α1,3GT but not from the we also that the recombinant α1,3GT in the culture media of cells not to in the was not These results demonstrate that the is cleaved from the of the recombinant α1,3GT from results the of secretion of the α1,3GT from we also which the large the full-length The media for α1,3GT enzyme and in the media was used for a the The media of and the of secreted α1,3GT from cells was in not which is to that of secreted α1,3GT from These that secretion of α1,3GT from cells is a which is that the enzyme is secreted from a large of cells S. K., Yeh, J.-C., and Cummings, R. D. (1997) Glycoconj. J. in press.Google Scholar). the of the α1,3GT secreted by the enzyme was from cell culture media by The enzyme was and to N-terminal as soluble the amino acid the The of the two reveal that soluble forms of α1,3GT are from the full-length form by cleavage C-terminal to and C-terminal to in the stem region of the as shown in A. We the of the secreted α1,3GT from which the N-terminal as The enzyme was from the culture media of the cells by and to which it an of The gel containing the enzyme was to N-terminal as two N-terminal sequences and these to in the soluble forms in the culture media from cells These results that for both the soluble and full-length forms of the α1,3GT specific proteolysis occurs within the sequence and These that proteolysis occurs within a region of the stem of the However, it is that cleavage sites also cleavage sites be N-terminal to the the two forms we a form of the full-length α1,3GT in which the stem sequence was to at sites for as shown in C. These by proteases that for The encoding was into 293 and from these cells by the Unexpectedly, the was cleaved as as the and the enzyme was in the culture media not The results comparable to shown in A. The of the enzyme is not These that proteolysis of the stem region in sites in to that the stem region in may a for a different These demonstrate that a soluble form of the α1,3GT, which is to that through proteolysis of the membrane-associated full-length is functional within the Golgi apparatus and is of of Galα1 → in cell and secreted glycoproteins. These the that soluble glycosyltransferases, by specific proteolysis of the membrane-associated enzymes within the in the biosynthesis of Numerous have the of soluble glycosyltransferases in (18Berger E.G. Kozdrowski I. Weiser M.M. van den Eijnden D.H. Schiphorst W.E. Eur. J. Biochem. 1978; 90: 213-222Google Scholar, 19Hudgin R.L. Schachter H. Can. J. Biochem. 1971; 49: 829-837Google Scholar, 23Debose-Boyd R.A. Nyame A.K. Smith D.F. Cummings R.D. Arch. Biochem. Biophys. 1996; 335: 109-117Google Scholar, J. C. C. J. 1978; 5: Scholar, A. J. Biol. Chem. 1981; Scholar, G. Jamieson J.C. Biochem. J. 1988; Scholar, J.C. W.E. R.L. J. Biol. Chem. Scholar, F. J. Biol. Chem. Scholar, F. Malagolini N. F. Arch. Biochem. Biophys. 1988; Scholar, Arch. Biochem. Biophys. Scholar) and in the growth media from normal and transformed cell lines W.D. Mastrangelo R. Weiser M.M. Cancer Res. 1981; 41: 2611-2615Google Scholar, 22Serafini-Cessi F. Malagolini N. Guerrini S. Turrini I. Glycoconj. J. 1995; 12: 773-779Google Scholar), although the functional of soluble enzymes has not been the mechanisms formation of soluble glycosyltransferases have not been It has been shown that soluble forms of the α2,6-sialyltransferase and the bovine β1,4-galactosyltransferase are by proteolysis within the stem domain (24D'Agostaro G. Bendiak B. Tropak M. Eur. J. Biochem. 1989; 183: 211-217Google Scholar, 25Weinstein J. Lee E.U. McEntee K. Lai P.-H. Paulson J.C. J. Biol. Chem. 1987; 262: 17735-17743Google Scholar). of the membrane-associated α2,6-sialyltransferase may involve a cathepsin D-like protease (26Lammers G. Jamieson J.C. Biochem. J. 1989; 261: 389-393Google Scholar) that containing T. K. H. H. J. Biochem. 1987; Scholar). an enzyme in cleavage of the α1,3GT, since to However, cleavage also in the which in by cathepsin D-like proteases These that cleavage sites in the stem region, in to we have defined, that the in sites for these lines be to the mechanisms of cleavage of the stem region of the α1,3GT and the proteases It has been that the α2,6-sialyltransferase is not secreted from cells K.J. Lee E.U. B. Paulson J.C. J. Biol. Chem. 1989; 264: Scholar), that cells a protease of the stem region of the we cells are of cleaving the stem domain of the murine α1,3GT and the of glycosyltransferases in cells may It has also been found that the human is not secreted from cells the cloned full-length cDNA R.D. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. Scholar), although is found in in human V.P. R.D. S. Lowe J.B. J. Biol. Chem. 1989; 264: Scholar, T. A. Proc. Natl. Acad. Sci. U. S. A. 1984; Scholar, J. R. J. 1985; Scholar). It is that secretion of a from cells is by a of the of the stem region and endogenous proteases in cells expressing the Numerous have the of the transmembrane and flanking sequences of glycosyltransferases for enzyme in the Golgi apparatus (6Teasdale R.D. D'Agostaro G. Gleeson P.A. J. Biol. Chem. 1992; 267: 4084-4096Google Scholar, 7Russo R.N. Shaper N.L. Taatjes D.J. Shaper J.H. J. Biol. Chem. 1992; 267: 9241-9247Google Scholar, 8Nilsson T. Lucoq J.M. Macay D. Warren G. EMBO J. 1991; 10: 3567-3575Google Scholar, 9Aoki D. Lee N. Yamaguchi N. Dubois C. Fukuda M.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4319-4323Google Scholar, 10Tang B.L. Wong S.H. Lowe S.H. Hong W. J. Biol. Chem. 1992; 267: 10122-10126Google Scholar, 11Burke J. Pettitt J.M. Humphris D. Gleeson P.A. J. Biol. Chem. 1994; 269: 12049-12059Google Scholar, 12Munro S. EMBO J. 1991; 10: 3577-3588Google Scholar, 13Colley K.J. Lee E.U. Paulson J.C. J Biol. Chem. 1992; 267: 7784-7793Google Scholar, 14Wong S.H. Low S.H. Hong W. J. Cell. Biol. 1992; 117: 245-258Google Scholar, 15Masibay A.S. Balaji P.V. Boeggeman E.E. Qasba P.K. J. Biol. Chem. 1993; 268: 9908-9916Google Scholar, 16Dahdal R.Y. Colley K.J. J. Biol. Chem. 1993; 268: 26310-26319Google Scholar, 17Munro S. EMBO J. 1995; 14: 4695-4705Google Scholar). However, in the transmembrane domain may not be for Golgi of and of the enzymes may also be soluble form of the membrane-associated form of in a T. C. R. F. G. Berger E.G. Warren G. EMBO J. 1994; Scholar). a membrane-associated enzyme as a for a soluble a soluble form of the α2,6-sialyltransferase containing the stem region and the catalytic domain is secreted from that the stem and/or the catalytic of that enzyme may to Golgi K.J. Lee E.U. Paulson J.C. J Biol. Chem. 1992; 267: 7784-7793Google Scholar). We that a of the soluble α1,3GT was in the cells and that it to be secreted. a for the α1,3GT and the enzyme important for Golgi are not but are in to these these results that a soluble be functional within the Golgi is the of the full-length It may be that the the soluble and full-length forms of the enzymes have different but The full-length forms of glycosyltransferases may be important in membrane-associated as and membrane-bound glycoproteins. We that the soluble α1,3GT was as efficient as the full-length enzyme in secreted proteins from 293 However, the soluble enzyme to be efficient the full-length enzyme in endogenous membrane-associated a may in the Golgi both the membrane-bound and soluble enzymes function to glycosylate the of The of secreted α1,3GT from cells was which is the of the cleaved two of However, the secreted α1,3GT from an of of the soluble the by proteolysis of the full-length enzyme in cells is that the of the be to be which is the The of the secreted enzyme from the full-length form may be to type of that occurs the full-length, membrane-associated a was also in β1,4-galactosyltransferase from human D.J. Biochem. Biophys. Res. Scholar). It has been that a soluble form of β1,4-galactosyltransferase the membrane-bound full-length β1,4-galactosyltransferase and that these to enzyme secretion (6Teasdale R.D. D'Agostaro G. Gleeson P.A. J. Biol. Chem. 1992; 267: 4084-4096Google Scholar). may in the full-length of α1,3GT as it through the and these may be different from that in the soluble form of the We are to the exact of the secreted α1,3GT from and the of the these have the of a soluble enzyme to glycosylate their biosynthesis within glycosyltransferases also function as soluble forms within cells to be in the it be to the of cleavage of the α1,3GT, by of the sequence in the stem region by of the protease(s) responsible for has an the of by the it also be important to the membrane-bound and soluble enzymes in their of of and of specific sites in We A. and W. the and the