The Structure and Assembly of Secreted Mucins
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.31751
ISSN1083-351X
AutoresJuan Pérez-Vilar, Robert L. Hill,
Tópico(s)Carbohydrate Chemistry and Synthesis
Resumoporcine submaxillary mucin bovine submaxillary mucin canine tracheobronchial mucin frog integumentary mucin mouse submandibular mucin porcine gastric mucin rat submandibular mucin human von Willebrand factor Mucins are major glycoprotein components of the mucous that coats the surfaces of cells lining the respiratory, digestive, and urogenital tracts, and in some amphibia, the skin. They function to protect epithelial cells from infection, dehydration, and physical or chemical injury, as well as to aid the passage of materials through a tract. Individual organisms make several structurally different mucins, and a given mucin may be found in more than one organ (see Supplemental Material). Members of the mucin family can differ considerably in size. Some are small, containing a few hundred amino acid residues, whereas others contain several thousands of residues and are among the largest known proteins. Irrespective of size, all mucin polypeptide chains have domains rich in threonine and/or serine whose hydroxyl groups are in O-glycosidic linkage with oligosaccharides. Moreover, these domains are composed of tandemly repeated sequences that vary in number, length, and amino acid sequence from one mucin to another (1Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 37: 607-634Crossref Scopus (854) Google Scholar). The carbohydrate content of a mucin may account for up to 90% of its weight. There are two types of mucins, membrane-bound and secreted. Of the human mucins, two are membrane-bound (MUC1 and MUC4) (2Gendler S.J. Lancaster C.A. Taylor-Papadimitriu J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E.N. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar, 3Moniaux N. Nollet S. Porchet N. Degand P. Laine A. Aubert J.-P. Biochem. J. 1999; 338: 325-333Crossref PubMed Scopus (219) Google Scholar) and four are secreted (MUC2, MUC5AC, MUC5B, and MUC7) (4Gum J.R. Hicks J.W. Toribara N.W. Siddiki B. Kim Y.S. J. Biol. Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar, 5Li D. Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Desseyn J.L. Aubert J.P. van Seuningen I. Porchet N. Laine A. J. Biol. Chem. 1997; 272: 16873-16883Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 7Bobek L.A. Tsa H. Biesbrock A.R. Levine M.J. J. Biol. Chem. 1993; 268: 20563-20569Abstract Full Text PDF PubMed Google Scholar). The three other mucins (MUC3, MUC6, and MUC8) (8Gum J.R. Ho J.J.L. Pratt W.S. Hicks J.W. Hill A.S. Vinall L.E. Roberton A.M. Swallow D.M. Kim Y.S. J. Biol. Chem. 1992; 267: 26678-26686Abstract Full Text PDF Google Scholar, 9Toribara N.W. Ho S.B. Gum E. Gum J.R. Lau P. Kim Y.S. J. Biol. Chem. 1997; 272: 16398-16403Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 10Shankar V. Pichan P. Eddy Jr., R.L. Tonk V. Nowak N. Sait S.N. Shows T.B. Schultz R.E. Gotway G. Elkins R.C. Gilmore M.S. Sachdev G.P. Am. J. Respir. Cell Mol. Biol. 1997; 16: 232-241Crossref PubMed Scopus (128) Google Scholar, 11Pigny P. Guyonnet-Duperat V. Hill A.S. Pratt W.S. Galiegue-Zouitina S. Vinall L. Collyn D'Hooge M. Laine A. Van-Seuningen I. Degand P. Gum J.R. Kim Y.S. Swallow D.M. Aubert J.-P. Porchet N. Genomics. 1996; 38: 340-352Crossref PubMed Scopus (187) Google Scholar) cannot be classified. Each human mucin has a counterpart in other animals. Thus, porcine submaxillary mucin (PSM)1 (12Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), one of the most thoroughly characterized mucins, has a tissue distribution and structure similar to MUC5B. An increasing number of proteins that are not mucins also contain highly O-glycosylated domains called “mucin-like domains.” The functions of mucins are dependent on their ability to form viscous solutions or gels. Although the highly glycosylated domains of mucins are devoid of secondary structures, they are long extended structures that are much less flexible than unglycosylated random coils. The oligosaccharides contribute to this stiffness in two ways, by limiting the rotation around peptide bonds and by charge repulsion among the neighboring, negatively charged oligosaccharide groups (13Jentoft N. Trends Biochem. Sci. 1991; 15: 291-294Abstract Full Text PDF Scopus (621) Google Scholar). Such long, extended molecules have a much greater solution volume than native or denatured proteins with little or no carbohydrate and endow aqueous mucin solutions with a high viscosity. Mucins protect against infection by microorganisms that bind cell surface carbohydrates, and mucin genes appear to be up-regulated by substances derived from bacteria, e.g. lipopolysaccharides (14Dohrman A. Miyata S. Gallup M. Li J.D. Chapelin C. Coste A. Escudier E. Nadel J. Basbaum C. Biochim. Biophys. Acta. 1998; 1406: 251-259Crossref PubMed Scopus (183) Google Scholar). This review will summarize what is known about the polypeptide structures of the secreted mucins and how some, in particular PSM, are assembled via interchain disulfide bonds into molecules with molecular weights in the millions. We will not consider membrane-bound mucins, which were the subject of earlier reviews (1Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 37: 607-634Crossref Scopus (854) Google Scholar, 15Hilkens J. Marjolyn J.L. Ligtenberg H.L.V. Litvinov S.V. Trends Biochem. Sci. 1992; 17: 359-363Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 16McNeer R.R. Huang D. Fregien N.L. Carraway K.L. Biochem. J. 1998; 330: 737-744Crossref PubMed Scopus (47) Google Scholar). Complete amino acid sequences have been described for frog (Xenopus) integumentary mucins FIM-A.1 (17Hoffmann W. J. Biol. Chem. 1988; 263: 7686-7690Abstract Full Text PDF PubMed Google Scholar) and FIM-B.1 (18Joba W. Hoffmann W. J. Biol. Chem. 1997; 272: 1805-1810Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), PSM (12Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), RSM (19Albone E.F. Hagen F.K. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1994; 269: 16845-16852Abstract Full Text PDF PubMed Google Scholar), MSM (20Denny P.C. Mirels L. Denny P.A. Glycobiology. 1996; 6: 43-50Crossref PubMed Scopus (28) Google Scholar), MUC2 (4Gum J.R. Hicks J.W. Toribara N.W. Siddiki B. Kim Y.S. J. Biol. Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar), MUC5B (6Desseyn J.L. Aubert J.P. van Seuningen I. Porchet N. Laine A. J. Biol. Chem. 1997; 272: 16873-16883Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), and MUC7 (7Bobek L.A. Tsa H. Biesbrock A.R. Levine M.J. J. Biol. Chem. 1993; 268: 20563-20569Abstract Full Text PDF PubMed Google Scholar) and almost complete sequences for MUC5AC (5Li D. Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and rat Muc2 (21Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar). The different domains of mucins are shown in Fig.1. Many of the domains show sequence identities and possibly similar functions in different mucins. These mucins vary greatly in size, from as few as 322 residues to 13,288 residues. The sequences of mucin polypeptides were deduced almost completely by recombinant DNA methods, and the physical-chemical properties of some mucins have not been determined. Nevertheless, it is well established that the oligosaccharides in many secreted mucins, e.g. PSM (22Gerken T.A. Owens C.L. Pasumarthy M. J. Biol. Chem. 1998; 273: 26580-26588Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), show structural microheterogeneity, with GalNAcα-O-Ser/Thr as the sugar-protein linkage upon which other sugars are added. Most mucins have negatively charged sugars, either sialic acid or O-sulfosaccharides. The number, length, and amino acid sequence of the repeats vary among different mucins, as shown in the Supplemental Material. The tandem repeat domains are flanked on either side by other types of domains (Fig. 1). All of the serine and threonine residues in the repeat domain of PSM have O-linked oligosaccharides (23Gerken T.A. Owens C.L. Pasumarthy M. J. Biol. Chem. 1997; 272: 9709-9719Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), but this is not known for other mucins. The repeats in some mucins have identical sequences, whereas in others the repeat sequence is degenerate. The lack of secondary structures in the repeat domains and their flanking domains suggests that these domains serve as a scaffold forO-linked oligosaccharides (24Eckhardt A.E. Timpte C.S. Abernethy J.L. Toumadje A. Johnson Jr., W.C. Hill R.L. J. Biol. Chem. 1987; 262: 11339-11344Abstract Full Text PDF PubMed Google Scholar), whose properties determine in large part the properties of a mucin. Light scattering and electron microscopy suggest that these glycosylated domains are semi-rigid, extended structures (13Jentoft N. Trends Biochem. Sci. 1991; 15: 291-294Abstract Full Text PDF Scopus (621) Google Scholar, 25Marianne T. Perini J.-M. Lafitte J.-J. Houdret N. Pruvot F.-R. Lamblin G. Slayter H.S. Roussel P. Biochem. J. 1987; 248: 189-195Crossref PubMed Scopus (31) Google Scholar). The tandem repeat domains in many mucins, e.g. PSM (12Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and MUC5B (6Desseyn J.L. Aubert J.P. van Seuningen I. Porchet N. Laine A. J. Biol. Chem. 1997; 272: 16873-16883Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), are encoded by a single large exon, although the remainder of the mucin is encoded by short exons separated by long introns. Many mucins show length polymorphism as the result of multiple alleles that encode different numbers of tandem repeats (26Vinall L.E. Hill A.S. Pigny P. Pratt W.S. Toribara N. Gum J.R. Kim Y.S. Porchet N. Aubert J.-P. Swallow D.M. Hum. Genet. 1998; 102: 357-366Crossref PubMed Scopus (70) Google Scholar). Thus, PSM is encoded by at least three alleles with 99, 110, and 135 repeats, respectively (12Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Several disulfide-rich domains are found in secreted mucins except RSM (19Albone E.F. Hagen F.K. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1994; 269: 16845-16852Abstract Full Text PDF PubMed Google Scholar), MSM (20Denny P.C. Mirels L. Denny P.A. Glycobiology. 1996; 6: 43-50Crossref PubMed Scopus (28) Google Scholar), and MUC7 (7Bobek L.A. Tsa H. Biesbrock A.R. Levine M.J. J. Biol. Chem. 1993; 268: 20563-20569Abstract Full Text PDF PubMed Google Scholar) and are often at either end of the polypeptide (Fig. 1). The disulfide-rich D-domain in mucins first found in VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar) is now recognized in many other proteins (28Kotani E. Yamakawa M. Iwamoto S. Tashiro M. Mori H. Sumida M. Matsubara F. Taniani K. Kadono-Okuda K. Kato Y. Mori H. Biochim. Biophys. Acta. 1995; 1260: 245-268Crossref PubMed Scopus (103) Google Scholar, 29Gao Z. Garbers D.L. J. Biol. Chem. 1998; 273: 3415-3421Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 30Cohen-Salmon M. El-Amraqui A. Leibovici M. Petit C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14450-14455Crossref PubMed Scopus (115) Google Scholar, 31Legan P.K. Rau A. Keen J.F. Richardson G.P. J. Biol. Chem. 1997; 272: 8791-8801Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Many secreted mucins contain three NH2-terminal D-domains, designated D1, D2, and D3, and some a fourth domain, D4, at the COOH terminus (Fig. 1). A partial D-domain, D′, is between D2 and D3 in all secreted mucins and VWF. Each domain, which contains up to 30 ½Cys, shows significant sequence identity with the other D-domains, especially the half-cystines. Comparisons of the sequences of the D-domains and other ½Cys-rich domains are given as supplemental information (see Supplemental Material). The D1-, D2-, and D3-domains of PSM areN-glycosylated when expressed in COS-7 cells (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), but this is not known for other mucins. In PSM and VWF all of the ½Cys in the D1-, D2-, D3-, and CK-domains are thought to form disulfide bonds, some of which are intrachain bonds whereas others are interchain bonds that are involved in assembly of PSM and VWF into multimers (see below). A 240–325-residue domain with 29–33 ½Cys is at the COOH terminus of many mucins (4Gum J.R. Hicks J.W. Toribara N.W. Siddiki B. Kim Y.S. J. Biol. Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar, 5Li D. Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Desseyn J.L. Aubert J.P. van Seuningen I. Porchet N. Laine A. J. Biol. Chem. 1997; 272: 16873-16883Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 12Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. Chem. 1997; 272: 33204-33210Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 18Joba W. Hoffmann W. J. Biol. Chem. 1997; 272: 1805-1810Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 33Jiang W. Woitach J.T. Keil R.L. Bhavanandan V.P. Biochem. J. 1998; 331: 193-199Crossref PubMed Scopus (16) Google Scholar, 34Verma M. Davidson E.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7144-7148Crossref PubMed Scopus (27) Google Scholar) (see Supplemental Material) (Fig. 1). These mucin domains have significant sequence identity with one another and with those at the carboxyl terminus of other proteins (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar, 28Kotani E. Yamakawa M. Iwamoto S. Tashiro M. Mori H. Sumida M. Matsubara F. Taniani K. Kadono-Okuda K. Kato Y. Mori H. Biochim. Biophys. Acta. 1995; 1260: 245-268Crossref PubMed Scopus (103) Google Scholar, 30Cohen-Salmon M. El-Amraqui A. Leibovici M. Petit C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14450-14455Crossref PubMed Scopus (115) Google Scholar). Like the D-domains, they are predicted to have globular structures with α-helices and pleated sheets and few or no free thiols (35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). They very likely contain N-linked oligosaccharides at one or more acceptor motifs (NX(S/T)) (36Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The first 100–130 residues in this domain have sequence identity with the C-domains of VWF, but the last 90–120 residues from the COOH terminus have sequence identities with the CK-domain at the COOH terminus of VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar). The CK-domains are homologous to the “cystine knot” superfamily of proteins that includes transforming growth factor β2, nerve growth factor, platelet-derived growth factor, and chorionic gonadotropin (37Sun P.D. Davies D.R. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 269-291Crossref PubMed Google Scholar). The CK-domains of VWF and mucins show significant sequence identity to norrin, a 133-residue protein that in mutant form gives rise to Norrie disease in humans, a rare, sex-linked disorder characterized by congenital blindness, mental retardation, and deafness (38Meitinger T. Meindl A. Bork P. Rost B. Sander C. Haasemann M. Murken J. Nat. Genet. 1993; 5: 376-380Crossref PubMed Scopus (151) Google Scholar). The CK-domain provides the ½Cys that form interchain disulfide bonds between the polypeptide chains of VWF and PSM and presumably other mucins (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar, 35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 36Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 39Bell S.L. Khatri I.A. Xu G. Forstner J.F. Eur. J. Biochem. 1998; 263: 123-131Crossref Scopus (33) Google Scholar) (see below). A B-domain with sequence identity to those in VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar) is found in several mucins (4Gum J.R. Hicks J.W. Toribara N.W. Siddiki B. Kim Y.S. J. Biol. Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar, 5Li D. Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Desseyn J.L. Aubert J.P. van Seuningen I. Porchet N. Laine A. J. Biol. Chem. 1997; 272: 16873-16883Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 21Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). Half-cystine-rich domains other than the D- and CK-domains are noted in a few mucins (Fig. 1), and P-domains like those in the trefoil factor family (40Hauser F. Hoffman W. J. Biol. Chem. 1992; 267: 24620-24624Abstract Full Text PDF PubMed Google Scholar, 41Sands B.E. Podolsky D.K. Annu. Rev. Physiol. 1996; 58: 253-273Crossref PubMed Scopus (198) Google Scholar) (see Supplemental Material) are in some frog mucins. Epidermal growth factor-like domains are found in other mucins (8Gum J.R. Ho J.J.L. Pratt W.S. Hicks J.W. Hill A.S. Vinall L.E. Roberton A.M. Swallow D.M. Kim Y.S. J. Biol. Chem. 1992; 267: 26678-26686Abstract Full Text PDF Google Scholar, 42Shekels L.L. Hunninghake D.A. Tisdale A.S. Gipson I.K. Kieliszewski M. Kozak C.A. Ho S.B. Biochem. J. 1998; 330: 1301-1308Crossref PubMed Scopus (48) Google Scholar,43Khatri I. Fostner G. Fostner J. Biochim. Biophys. Acta. 1997; 1326: 7-11Crossref PubMed Scopus (24) Google Scholar). It is well known that the molecular weight of many mucins decreases in the presence of reducing agents (e.g. Ref. 44Shogren R.L. Jamieson A.M. Blackwell J. Jentoft N. J. Biol. Chem. 1984; 259: 14657-14662Abstract Full Text PDF PubMed Google Scholar), suggesting that interchain disulfide bonds maintain mucins in a multimeric state. Studies on the biosynthesis of mucins in tissue explants (45Strous G.J. Dekker J. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 57-92Crossref PubMed Scopus (752) Google Scholar) and cells in culture (46McCool D.J. Forstner G. Forstner J. Biochem. J. 1994; 302: 111-118Crossref PubMed Scopus (53) Google Scholar, 47Sheehan J.K. Thornton D.J. Howard M. Carlstedt I. Corfield A.P. Pareskeva C. Biochem. J. 1996; 315: 1055-1060Crossref PubMed Scopus (38) Google Scholar, 48Van Klinken B.J.-W. Einerhand A.W.C. Buller H.A. Dekker J. Glycobiology. 1998; 8: 67-75Crossref PubMed Scopus (55) Google Scholar, 49Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. J. Biol. Chem. 1998; 273: 18857-18863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 50Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. Biochem. J. 1998; 335: 381-387Crossref PubMed Scopus (37) Google Scholar) have confirmed the role of disulfide bonds in the assembly of mucins into multimers. The recognition that mucins had disulfide-rich domains structurally similar to those in VWF and the fact that VWF formed disulfide-bonded multimers through its disulfide-rich domains (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar) indicated a possible role of these domains in mucin multimer formation. However, the large size of mucin polypeptides and their high carbohydrate content prevented use of the conventional methods of protein chemistry for examining the molecular details of mucin multimer formation. Fortunately it has been possible to obtain insights into multimer formation by expression of plasmids encoding mucin domains in mammalian cells followed by characterization of the recombinant proteins by SDS-gel electrophoresis and chromatography under reducing and non-reducing conditions. This approach has been particularly successful for examining multimer formation in PSM (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 36Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar), with the assumption that the assembly of domains accurately reflects the assembly of native mucinsin vivo. Thus, as illustrated in Fig.2, PSM is thought to form disulfide-linked dimers through its COOH-terminal CK-domains, and the dimers then form disulfide-bonded multimers through their NH2-terminal D-domains. It is likely that all mucins structurally related to VWF (Fig. 1), in addition to rat Muc2, MUC5AC, BSM, CTM, PGM, and MUC6, form multimers similar to those formed by PSM. Two polypeptide chains of PSM form disulfide-linked dimers through their CK-domains soon after their biosynthesis in the endoplasmic reticulum (35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 36Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Pulse-chase studies show that dimerization is very rapid and occurs concomitant with or soon after N-glycosylation. N-Glycosylation is not required for dimer formation or later during multimer formation because both processes are unaffected by tunicamycin (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 36Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, unglycosylated species are poorly secreted and/or rapidly degraded after secretion into the extracellular medium (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The fact that brefeldin A, a compound that disrupts the Golgi complex, has no effect on dimer formation and that dimers are formed before N-linked oligosaccharides become endoglycosidase H-resistant indicates that dimerization is confined to the endoplasmic reticulum. Subsequent to the studies on the dimerization of PSM, rat Muc2 was also reported to form disulfide-linked dimers through its COOH-terminal disulfide-rich domain, which includes the CK-domain (39Bell S.L. Khatri I.A. Xu G. Forstner J.F. Eur. J. Biochem. 1998; 263: 123-131Crossref Scopus (33) Google Scholar) (see Supplemental Material). Dimer formation by other types of mucins has not been examined by expression of plasmids encoding the CK-domains. However, mucins secreted by mucin-producing cells in culture (47Sheehan J.K. Thornton D.J. Howard M. Carlstedt I. Corfield A.P. Pareskeva C. Biochem. J. 1996; 315: 1055-1060Crossref PubMed Scopus (38) Google Scholar, 48Van Klinken B.J.-W. Einerhand A.W.C. Buller H.A. Dekker J. Glycobiology. 1998; 8: 67-75Crossref PubMed Scopus (55) Google Scholar, 49Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. J. Biol. Chem. 1998; 273: 18857-18863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 50Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. Biochem. J. 1998; 335: 381-387Crossref PubMed Scopus (37) Google Scholar), including MUC2, MUC5AC, and likely MUC5B and MUC6, appear to form disulfide-linked dimers shortly after their synthesis in the endoplasmic reticulum. In contrast to PSM, N-glycosylation is reported to be required for dimerization of rat Muc2, MUC2, and MUC5AC. The interchain disulfide bonds in PSM dimers have been examined by site-directed mutagenesis (35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Of the 11 ½Cys in the CK-domain, mutation of 8 is without effect on dimer formation. Dimerization is partly impaired by mutation of 3 ½Cys at residues 13223, 13244, and 13246. C13244 and C13246 are in the sequence C13244LC13246C, which is conserved in all mucins and other proteins containing the CK-domain (Fig.3) (see Supplemental Material) and is also critical for interchain disulfide bond formation in VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar) and norrin (53Perez-Vilar J. Hill R.L. J. Biol. Chem. 1997; 272: 33410-33415Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). C13223 in PSM in the sequence C13223VGEC is also required for efficient dimer formation, but the mutant proteins at this residue are poorly secreted (35Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), suggesting that this sequence motif may be important in folding of the CK-domain in the endoplasmic reticulum. This sequence motif is also conserved in all mucins, VWF, and norrin (Fig. 3) (see Supplemental Material), which attests to its importance in maintaining the structure of the CK-domain. The incorporation ofO-linked oligosaccharides into mucins begins afterN-glycosylation and disulfide-linked dimer formation as suggested by biosynthetic studies on MUC2 (49Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. J. Biol. Chem. 1998; 273: 18857-18863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and MUC5AC (50Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. Biochem. J. 1998; 335: 381-387Crossref PubMed Scopus (37) Google Scholar) and cytochemical studies of PSM (54Roth J. Wang Y. Eckhardt A.E. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8935-8939Crossref PubMed Scopus (115) Google Scholar, 55Deschuyteneer M. Eckhardt A.E. Roth J. Hill R.L. J. Biol. Chem. 1988; 263: 2452-2459Abstract Full Text PDF PubMed Google Scholar). O-Glycosylation of PSM begins when the dimers reach the cis-Golgi compartments, because the GalNAc transferase that forms the GalNAc-Ser/Thr linkages and the mucin precursors bearing only GalNAc have been located by electron microscopy in the cis-Golgi in mucous cells of submaxillary glands (54Roth J. Wang Y. Eckhardt A.E. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8935-8939Crossref PubMed Scopus (115) Google Scholar, 55Deschuyteneer M. Eckhardt A.E. Roth J. Hill R.L. J. Biol. Chem. 1988; 263: 2452-2459Abstract Full Text PDF PubMed Google Scholar). Moreover, unglycosylated mucin precursors are detected only in the lumen of the endoplasmic reticulum (55Deschuyteneer M. Eckhardt A.E. Roth J. Hill R.L. J. Biol. Chem. 1988; 263: 2452-2459Abstract Full Text PDF PubMed Google Scholar). Other cells expressing secreted mucins, including intestinal goblet cells (56Roth J. J. Cell Biol. 1984; 98: 399-406Crossref PubMed Scopus (148) Google Scholar), also appear to initiate O-glycosylation in thecis-Golgi although in certain mucin-producing cell linesO-glycosylation is found to begin in the endoplasmic reticulum (46McCool D.J. Forstner G. Forstner J. Biochem. J. 1994; 302: 111-118Crossref PubMed Scopus (53) Google Scholar, 57Perez-Vilar J. Hidalgo J. Velasco A. J. Biol. Chem. 1991; 266: 23967-23976Abstract Full Text PDF PubMed Google Scholar). The completion of the biosynthesis of theO-linked oligosaccharides in secreted mucins continues in the medial- and trans-Golgi compartments where the requisite glycosyltransferases for elongation and termination of the oligosaccharides are located (58Van Den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-168Crossref PubMed Scopus (602) Google Scholar). Expression in COS-7 cells of plasmids encoding the three D-domains of PSM has shown that these domains participate in formation of interchain disulfide bonds between disulfide-linked dimers to give very high molecular weight multimers of mucin (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Multimer formation differs from dimer formation in several respects. Brefeldin A, which disrupts the Golgi complex, inhibits multimer formation, indicating that multimers form in the Golgi complex. Compounds that increase the pH of thetrans-Golgi compartments, such as chloroquine and monensin, also inhibit multimer formation but not dimerization (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Bafilomycin, a specific inhibitor of the vacuolar H+-ATPase that maintains the trans-Golgi compartments at a slightly acidic pH, also inhibits multimer formation. These observations suggest that the interchain disulfide bonds that give rise to multimers are formed at a slightly acidic pH in the trans-Golgi complex through ½Cys residues in the D-domains. The molecular weights of the multimers cannot be assessed accurately by SDS-gel electrophoresis because they are so large they do not enter the running gel under non-reducing conditions. However, species with a size of trimers were observed when the three D-domains were expressed together (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), suggesting that a step in the process of multimerization is trimer formation of disulfide-linked dimers. Such multimers are likely branched structures as indicated in Fig. 2. Recombinant PSM containing no glycosylated domains is secreted from COS-7 cells as dimers and multimers and indicates that like VWF not all dimers are converted to multimers (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). C1199 in the D3-domain of PSM has been found by site-directed mutagenesis studies to be a likely candidate for forming one of the interchain disulfide bonds in mucin multimers (51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar). It is in the sequence C1199SWRYEPCG, which is highly conserved in secreted mucins (Fig. 3) (see Supplemental Material), and the analogous ½Cys in VWF is required for its multimerization (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar). In contrast, C1276, which is suggested to form interchain disulfide bonds in VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar), does not form such bonds in PSM (51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar). The other half-cystines in the D-domains that form interchain disulfide bonds are not known. VWF multimers are formed from prepro-VWF, which is cleaved intracellularly by a subtilisin-like protease (furin) at R763 in the sequence motif R760SKR763 in the D′-domain (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar). The released propeptide contains the D1- and D2-domains and is essential for multimer formation although cleavage is not. Cleavage may not be essential for mucin multimerization because the D′-domains of mucins do not contain the sequence motif required for proteolytic cleavage of prepro-VWF. The observation that the D-domains of PSM are not cleaved when expressed in COS-7 or MOP-8 cells (32Perez-Vilar J. Eckhardt A. DeLuca A.W. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) is consistent with the lack of the cleavage motif in the D′-domain of PSM (see Supplemental Material). However, some proteolytic processing of mucins is possible as suggested by recent studies showing that cleavage occurs in the COOH-terminal region of MUC2 (59Herrmann A. Davies J.R. Lindell G. Martensson S. Packer N.H. Swallow D.M. Carlstedt I. J. Biol. Chem. 1999; 274: 15828-15836Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), MUC5B (60Wickstrom C. Davies J.R. Eriksen G.V. Veerman E.C.I. Carlstedt I. Biochem. J. 1998; 334: 685-693Crossref PubMed Scopus (268) Google Scholar), and rat Muc2 (61Gongqiao X.G. Forstner G.G. Forstner J.F. Glycoconjugate J. 1996; 13: 81-90Crossref PubMed Scopus (8) Google Scholar), although a role for such cleavages in mucin assembly is unknown. Moreover, proteolytic cleavage during purification of the mucins was not ruled out. The prediction that PSM contains branched multimers indicates that branches should be observed on electron microscopy of mucins. It is generally argued that mucins form linear polymers (e.g. see Ref. 45Strous G.J. Dekker J. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 57-92Crossref PubMed Scopus (752) Google Scholar), which has been substantiated by electron microscopy (24Eckhardt A.E. Timpte C.S. Abernethy J.L. Toumadje A. Johnson Jr., W.C. Hill R.L. J. Biol. Chem. 1987; 262: 11339-11344Abstract Full Text PDF PubMed Google Scholar). Moreover, VWF forms dimers through its CK-domains, and the dimers form linear multimers through their d-domains (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar). It is quite possible that some mucins with disulfide-rich domains do not form multimers in the manner proposed for PSM. However, mucins are highly susceptible to proteolysis during isolation (62Eckhardt A.E. Timpte C.S. Abernethy J.L. Zhao Y. Hill R.L. J. Biol. Chem. 1991; 266: 9678-9686Abstract Full Text PDF PubMed Google Scholar, 63Khatri I.A. Forstner G.G. Forstner J.F. Biochem. J. 1998; 331: 323-330Crossref PubMed Scopus (21) Google Scholar), and further electron microscopic studies should be made on well characterized preparations. Of interest is a recent report describing branched structures for MUC5B in respiratory secretions of asthmatic individuals (64Sheehan J.K. Howard M. Richardson P.S. Longwill T. Thornton D.J. Biochem. J. 1999; 338: 507-513Crossref PubMed Scopus (91) Google Scholar). Nevertheless, additional mechanisms of mucin assembly are supported by studies on MUC2. LS174T cells synthesize soluble MUC2 disulfide-linked dimers, but higher molecular weight species are water-insoluble (65Axelsson M.A.B. Asker N. Hansson G.C. J. Biol. Chem. 1998; 273: 18864-18870Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Apparently, the water-insoluble species are assembled in the Golgi complex following initialO-glycosylation by a pH-independent process. These insoluble complexes are partly maintained by non-reducible chemical bonds of unknown nature (65Axelsson M.A.B. Asker N. Hansson G.C. J. Biol. Chem. 1998; 273: 18864-18870Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It is not known whether the complexes are secreted, although MUC2 in the intestine is thought to be part of an insoluble glycoprotein complex (59Herrmann A. Davies J.R. Lindell G. Martensson S. Packer N.H. Swallow D.M. Carlstedt I. J. Biol. Chem. 1999; 274: 15828-15836Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Other studies suggest that MUC2 is assembled into large soluble, disulfide-linked oligomers/multimers (47Sheehan J.K. Thornton D.J. Howard M. Carlstedt I. Corfield A.P. Pareskeva C. Biochem. J. 1996; 315: 1055-1060Crossref PubMed Scopus (38) Google Scholar,66Aksoy N. Thornton D.J. Corfield A. Paraskeva C. Sheehan J.K. Glycobiology. 1999; 9: 739-746Crossref PubMed Scopus (32) Google Scholar). Clearly, MUC5AC (67Hovenberg H.W. Davies J.R. Carlstedt I. Biochem. J. 1996; 318: 319-324Crossref PubMed Scopus (243) Google Scholar) and MUC5B (60Wickstrom C. Davies J.R. Eriksen G.V. Veerman E.C.I. Carlstedt I. Biochem. J. 1998; 334: 685-693Crossref PubMed Scopus (268) Google Scholar) are large soluble, gel-forming mucins stabilized by disulfide bonds. As described above, the assembly of PSM and VWF (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar) involves dimerization in the endoplasmic reticulum and multimerization in the trans-Golgi compartments. The molecular mechanisms that permit this compartmentalization are not known, but the NH2-terminal D-domains and the CGLCG motifs in the D1- and D3-domains seem to play critical roles (51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar). Plasmids encoding only the D1- and D2-domains, the D1- and the D3-domains, or the D3-domain of PSM expressed mucin oligomers in the presence of monensin suggesting that the three domains must be contiguous to avoid multimerization at the non-acidic pH of the endoplasmic reticulum and the cis- andmedial-Golgi compartments. Replacement of the two ½Cys by alanine in the CGLCG motif in the D3-domain permits formation of multimers in the presence of monensin (51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar). Thus, the motif in the D3-domain prevents multimerization of mucin in the non-acidic compartments of the endoplasmic reticulum and thecis/medial-Golgi compartments. Replacement of the two ½Cys by alanine in the CGLCG motif in the D1-domain dramatically reduces the rate of formation of disulfide-linked multimers (51Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 33527-33534Google Scholar). This observation suggests that multimerization at low pH in the acidic trans-Golgi compartments requires the motif in the D1-domain. Multimerization of VWF also requires the CGLCG motif in the D1-domain (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar), although a role for the motif in the D3-domain has not been reported. VWF has another CGLCG motif in the D2-domain that is also required for its assembly (27Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1109) Google Scholar). However, among the mucins structurally related to VWF, only MUC5AC and MUC5B have CGLCG motifs in their D2-domains (see Supplemental Material). The exact roles of the CGLCG motifs remain unknown but because of the fact that similar motifs are in the active sites of proteins involved in catalyzing formation of disulfide bonds during protein folding, such as protein disulfide isomerase (Fig. 3), the question arises whether these motifs have a direct role in formation of disulfide bonds in mucins. Much progress has been made recently in our understanding of the structure and assembly of secretory mucins, but much work remains for the future. Other members of the mucin family should be identified and their structures and mechanism of assembly into disulfide-bonded multimers elucidated. The pairing of half-cystines to form the many disulfide bonds in the globular domains must be established, and the role of chaperones in folding of these domains must also be determined. The molecular basis for the regulated/polarized transport of mucins should be explored. These kinds of studies will be needed to obtain further insights into the exact biological roles of mucins. Download .pdf (3.43 MB) Help with pdf files
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