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Identification of the Half-cystine Residues in Porcine Submaxillary Mucin Critical for Multimerization through the D-domains

1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês

10.1074/jbc.273.51.34527

1083-351X

Juan Pérez-Vilar, Robert L. Hill,

Skin and Cellular Biology Research

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin. Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin. Secretory mucins are synthesized by epithelial cells that line the luminal surfaces of the respiratory, gastrointestinal, and urogenital tracts of vertebrates, where they serve to protect the cells from physical injury, dehydration, and microbial infection. They are also found on the skin of amphibia. All of these glycoproteins have in common a central domain containing multiple, tandemly repeated sequences rich in serine and threonine to which O-linked oligosaccharides are attached. However, the number, length, and amino acid sequences of the repeats vary among different mucins (reviewed in Ref. 1Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 37: 607-634Crossref Scopus (872) Google Scholar). One group of secreted mucins, including porcine submaxillary mucin 1GenBankTM accession numberAF005273. (2Timpte C.S. Eckhardt A.E. Abernethy J.L. Hill R.L. J. Biol. Chem. 1988; 263: 1081-1088Abstract Full Text PDF PubMed Google Scholar, 3Eckhardt 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, 4Eckhardt A.E. Timpte C.S. DeLuca A.W. Hill R.L. J. Biol. 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Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar) and its rat homologue rMuc2 4GenBankTM accession numberM76740. (11Xu G. Huan L.-J. Khatri I.A. Wang D. Bennick A. Fahim R.E.F. Fostner G.G. Fostner J.F. J. Biol. Chem. 1992; 267: 5401-5407Abstract Full Text PDF PubMed Google Scholar, 12Carlstedt I. Herrmann A. Karlsson H. Sheehan J. Fransson L.-A. Hansson G.C. J. Biol. Chem. 1993; 268: 18771-18781Abstract Full Text PDF PubMed Google Scholar, 13Hansson G.C. Baeckstrom D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar, 14Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum Jr., J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar), and human mucin MUC5AC 5GenBankTM accession numberAF015521. (15Meerzaman D. Charles P. Daskal E. Polymeropoulus M.H. Martin B.M. Rose M.C. J. Biol. Chem. 1994; 269: 12932-12939Abstract Full Text PDF PubMed Google Scholar, 16Lesuffleur T. Roche F. Hill A.S. Lacasa M. Fox M. Swallow D.M. Zweibaum A. Real F.X. J. Biol. Chem. 1995; 270: 13665-13673Crossref PubMed Scopus (88) Google Scholar, 17Klomp L.W.J. VanRens L. Strous G.J. Biochem. J. 1995; 308: 831-838Crossref PubMed Scopus (45) Google Scholar, 18Li 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), has analogous half-cystine-rich domains at their amino and carboxyl termini. The roles of these disulfide-rich domains in the assembly of porcine submaxillary mucin into multimers linked by disulfide bonds have been reported earlier (19Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 20Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The similarity in the domain structures of many of the secreted mucins suggests that they share similar mechanisms of assembly. Porcine submaxillary mucin contains multiple tandem repeats, 81 residues in length with identical amino acid sequences (4Eckhardt 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). The polypeptide backbone of submaxillary mucin is encoded by one polymorphic gene with at least three alleles, each encoding different numbers of repeats (90, 125, and 133, respectively) (4Eckhardt 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). The polypeptide contains up to 13,288 residues (4Eckhardt 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), with a disulfide-rich region at its amino terminus containing the D1-, D2-, and D3-domains first identified in human prepro-von Willebrand factor 6GenBankTM accession numberX04385. (22Sadler J.E. J. Biol. Chem. 1991; 266: 22777-22780Abstract Full Text PDF PubMed Google Scholar) and human mucin MUC2 (10Gum Jr., 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), but now recognized to occur in many proteins, including other secreted mucins, Bombyx morihemocytin 7GenBankTM accession numberD29738. (23Kotani 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 (106) Google Scholar), mouse α-tectorin 8GenBankTM accession numberX99805. (24Legan P.K. Rau A. Keen J.F. Richardson G.P. J. Biol. Chem. 1997; 272: 8791-8801Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), mouse 9GenBankTM accession numberU97068. (25Gao Z. Garbers D.L. J. Biol. Chem. 1998; 273: 3415-3421Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and porcine 10GenBankTM accession numberU40024. (26Hardy D.M. Garbers D.L. J. Biol. Chem. 1995; 270: 26025-26028Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) zonadhesins, and mouse otogelin 11GenbankTM accession numberU96411. (27Cohen-Salmon M. El-Amraqui A. Leibovici M. Petit C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14450-14455Crossref PubMed Scopus (119) Google Scholar). A 240-residue half-cystine-rich domain is at the carboxyl terminus of porcine mucin, and the half-cystines in this domain are in positions corresponding to the half-cystines in carboxyl-terminal disulfide-rich domains of prepro-von Willebrand factor, hemocytin, otogelin, and the secreted mucins listed above. Porcine submaxillary mucin is N-glycosylated and forms disulfide-linked dimers through its carboxyl-terminal disulfide-rich domains while in the endoplasmic reticulum (19Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Only the most carboxyl-terminal 90 residues in the mucin including 11 half-cystines are involved in dimer formation (20Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The half-cystines in the 90-residue carboxyl-terminal sequence are in positions corresponding to those in Norrie disease protein (norrin), a 133-residue protein that forms disulfide-linked dimers and oligomers (28Perez-Vilar J. Hill R.L. J. Biol. Chem. 1997; 272: 33410-33415Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). One or more of the half-cystine residues at positions 13223, 13244, and 13246 in the norrin-like domain are involved in dimer formation of submaxillary mucin (20Perez-Vilar J. Hill R.L. J. Biol. Chem. 1998; 273: 6982-6988Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Mucin dimers areO-glycosylated in the Golgi complex (29Deschuyteneer M. Eckhardt A.E. Roth J. Hill R.L. J. Biol. Chem. 1988; 263: 2452-2459Abstract Full Text PDF PubMed Google Scholar, 30Roth 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) and, upon reaching the trans-Golgi compartments, are assembled into disulfide-bonded multimers via disulfide-bonded trimers of the amino-terminal D-domains (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Unlike human prepro-von Willebrand factor (31Ruggeri Z.M. Ware J. FASEB J. 1993; 7: 308-316Crossref PubMed Scopus (273) Google Scholar), there is no proteolysis of the mucin D-domains by furin prior to secretion (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Mucin multimerization appears to occur at a slightly acidic pH maintained by vacuolar H+-ATPase in the trans-Golgi compartments (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Although the covalent assembly of other mucins has not been established to any great extent, rat rMuc2 is reported to form disulfide-linked dimers through its carboxyl-terminal disulfide-rich domains (32Bell S.L. Khatri I.A. Xu G. Fostner J.F. Eur. J. Biochem. 1998; 253: 123-131Crossref PubMed Scopus (33) Google Scholar), and human MUC2 synthesized by LS174T cells forms disulfide-linked dimers while in the endoplasmic reticulum (33Van Klinken B.J.-W. Einerhand A.W.C. Buller H.A. Dekker J. Glycobiology. 1998; 8: 67-75Crossref PubMed Scopus (56) Google Scholar,34Asker 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 (109) Google Scholar). To obtain further insight into the half-cystines involved in forming disulfide-linked multimers, the proteins expressed by cells transfected with plasmids encoding half-cystine mutants of the amino-terminal region of porcine submaxillary mucin have been examined. This study has identified half-cystine 1199 and the two half-cystines in the CGLCG motif in the D1-domain to be involved in forming multimers and the two half-cystines in the CGLCG motif in the D3-domain to impede formation of multimers of mucin in non-acidic compartments. The expression vector pSMD1D2H was prepared from pMNH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), which encodes the entire amino-terminal region containing the D1-, D2-, and D3-domains of submaxillary mucin (residues 1–1360) plus a six-histidine tag at its carboxyl terminus. pMNH was digested with PstI and religated to create pMD1D2H, which was digested with NheI andXhoI to give a DNA fragment that was subcloned into the pSI plasmid (Promega) to create pSMD1D2H. This vector encodes the complete D1- and D2-domains of mucin (residues 1–835) followed by mucin residues 1172–1360, including a truncated D3-domain (residues 1172–1280) and the sequence GGRHHHHHH at the carboxyl terminus. The pMD1D3H vector was made by digestion of pMNH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) with EcoRI and BstXI and ligation of the resulting 3.6-kilobase pair DNA and the opened plasmid. The new vector was digested withNheI and XhoI, and the resulting DNA fragment was subcloned into pSI to give pSMD1D3H, which encodes residues 1–524 of submaxillary mucin, including the signal peptide and the complete D1-domain (residues 130–465), followed by residues 875–1360, which include the entire D3-domain (residues 925–1280) and the sequence GGRHHHHHH at the carboxyl terminus. The DNA that encodes the D1- and D2-domains was removed from pMNH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) by EcoRI digestion and religation of the open vector to give pMND3H.1. A DNA encoding the signal peptide (residues 1–36) of submaxillary mucin was made by DNA polymerase chain reaction withTaq DNA polymerase (Life Technologies, Inc.), the pMN vector (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as template, and the following primers (Life Technologies, Inc.): 5′-TAATACGACTCACTATAGGG-3′ and 5′-TATGAATTCTCTGATTTTAACCTGAAGAATAGGGCTTCCCC-3′. The amplified DNA was digested with KpnI and EcoRI and subcloned into pMND3H.1. The new plasmid was digested with NheI andXhoI, and the resulting DNA was subcloned into the pSI plasmid to give pSMD3H. This plasmid encodes a fusion protein containing the signal peptide (residues 1–34) and residues 876–1360 of submaxillary mucin, including the D3-domain (residues 925–1280) and the sequence GGRHHHHHH at its carboxyl terminus. Plasmid pSMD3CH was made by subcloning mucin DNA that was obtained by digesting pSMNCH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) with ApaI and SalI between the corresponding sites of plasmid pSMD3H. The new vector encodes a fusion protein containing the mucin signal peptide, the entire D3-domain (residues 925–1280), the complete carboxyl-terminal disulfide-rich domain (residues 13045–13288), and a six-histidine tag at the carboxyl terminus. The nucleotide sequences of all plasmids were verified by restriction analysis, partial DNA sequencing with Sequenase 2.0 (U. S. Biochemical Corp.), and in vitro transcription/translation assays with the T7-TNT coupled system (Promega) as directed by the manufacturer. Mutant plasmids were made as described earlier (35Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar) using the vector pSMNH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as template and the selection oligonucleotide 5′-GGTACCTCTAGTGTCGACCCGG-3′ (Life Technologies, Inc.), which destroys the unique XbaI site of pSMNH. The mutagenic oligonucleotides (Life Technologies, Inc.) were as follows: C1199A, 5′-GCACCTGGGGAAGCTAGCTGGCGTTATG-3′; C1276A, 5′-GATCCAGGATAACGCCGGGAGAATATGC-3′; C253A/C256A, 5′-CACAAACAATATCCAACTGCAGGTCTTGCTGGTAACTTCAACAATACTCC-3′; and C1058A/C1061A, 5′-TGGAATGGCAAAGTGGCCGGCCTTGCCGGAAACAATAATGGCG-3′. To facilitate screening, all mutagenic oligonucleotides were designed to produce silent mutations that destroy or create restriction sites in the DNA encoding mucin along with the desired codon changes. The nucleotide sequences of mutants were verified by restriction analysis, DNA sequencing, and in vitro transcription/translation assays. COS-7 cells were grown, maintained, and transfected with expression vectors with Fugene-6 (Boehringer Mannheim) as reported earlier (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Antiserum 5287 against the amino-terminal region of mucin (residues 1059–1360) was also described earlier (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Unless otherwise indicated, metabolic labeling of cells with [35S]cysteine (ICN), purification of the recombinant proteins by either immunoprecipitation with antiserum 5287 or absorption on TALON-IMAC beads (CLONTECH), and analysis of the proteins by SDS-gel electrophoresis and autoradiography were as described previously (19Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In some experiments, immunoprecipitates obtained with antiserum 5287 were solubilized with 50 mm sodium phosphate, pH 8.0, containing 10 mm Tris-HCl, pH 8.0, 0.1 m NaCl, and 6 m guanidine HCl, pH 8.0, and absorbed on TALON-IMAC beads as described earlier (28Perez-Vilar J. Hill R.L. J. Biol. Chem. 1997; 272: 33410-33415Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 14C-Methylated proteins (Amersham Pharmacia Biotech) used as molecular weight standards were myosin (220,000), phosphorylase b (97,000–100,000), bovine serum albumin (66,000), ovalbumin (46,000–50,000), carbonic anhydrase (30,000), and lysozyme (14,300). Half-cystine 1142 (36Dong Z. Thoma R.S. Crimmins D.L. McCourt D.W. Tuley E.A. Sadler J.E. J. Biol. Chem. 1994; 269: 6753-6758Abstract Full Text PDF PubMed Google Scholar) and half-cystine 1225 (37Azuma H. Hayashi T. Dent J.A. Ruggeri Z.M. Ware J. J. Biol. Chem. 1993; 268: 2821-2827Abstract Full Text PDF PubMed Google Scholar) have been suggested to form interchain disulfide bonds in multimers of human prepro-von Willebrand factor. The corresponding half-cystines in porcine submaxillary mucin are half-cystines 1199 and 1276, which are located in the amino-terminal D3-domain (Fig. 1). The codons for these half-cystines were changed one at a time by site-specific mutagenesis to alanine codons in plasmid pSMNH (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), which encodes the three D-domains of mucin plus a His tag (Fig. 1). The mutant plasmids (C1199A and C1276A) were then transiently expressed in COS-7 cells, and 48 h after transfection, the cells were incubated in medium containing [35S]cysteine. Radiolabeled proteins from the medium were purified on TALON-IMAC beads and then analyzed by SDS-gel electrophoresis and autoradiography. As shown in Fig.2, both mutant plasmids expressed a single protein with M r ∼ 200,000 on reducing SDS-gel electrophoresis (lanes 2 and3). These proteins were indistinguishable from that expressed by unmutated plasmid (lane 1), which, as shown earlier (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), corresponds to reduced N-glycosylated monomers of the amino-terminal region of mucin based on its sensitivity toN-glycanase and tunicamycin. These results show that secretion and likely N-glycosylation of the mucin amino-terminal region were not altered by either mutation.Figure 2Expression and secretion of mutants C1199A and C1276A of the amino-terminal region of mucin. COS-7 cells transfected with the expression vector pSMNH (lanes 1 and 4), C1199A (lanes 2and 5), or C1276A (lanes 3 and6) were metabolically labeled 48 h after transfection with [35S]cysteine for 4 h. Proteins from the medium were purified by absorption on TALON-IMAC beads and analyzed by SDS-gel electrophoresis and autoradiography after reduction in 2-mercaptoethanol (lanes 1–3) or without reduction (lanes 4–6). M andT indicate the positions of monomers and trimers, respectively, in unreduced samples. Top is the interface between the stacking and running gels. The molecular weights (MW) of the standards are in thousands.View Large Image Figure ViewerDownload (PPT) Fig. 2 also shows the protein species expressed by cells transfected with the mutant plasmids when analyzed on nonreducing gels. As shown earlier (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), unmutated plasmid pSMNH (lane 4) produced monomers (M r ∼ 180,000) and a second, slow migrating disulfide-linked oligomeric form that enters the resolving gel. On SDS-gel electrophoresis, this oligomeric form, also as shown earlier (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), has a M r of ∼520,000 and migrates slower than dimeric fibronectin (M r ∼ 450,000) and faster than unreduced thyroglobulin (M r ∼ 660,000), which is consistent with disulfide-linked trimers. Mutant C1199A (lane 5) expressed almost entirely monomers (M r ∼ 180,000) and only small amounts of species the size of trimers. In contrast, mutant C1276A (lane 6) expressed both monomers and trimers similar in size to those produced by unmutated plasmid pSMNH (lane 4). Since multimerization of submaxillary mucin occurs by dimerization of the carboxyl-terminal disulfide-rich domains and subsequent oligomerization through the amino-terminal D-domains (19Perez-Vilar J. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1996; 271: 9845-9850Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), these observations suggest that half-cystine 1199, but not half-cystine 1276, is involved in formation of disulfide-linked multimers through the D-domains of the mucin. To determine the role of the three D-domains in mucin multimerization, three different plasmids were prepared and expressed in COS-7 cells. As shown schematically in Fig.1, plasmid pSMD1D2H is devoid of the D3-domain and encodes the D1- and D2-domains with a carboxyl-terminal His tag. Plasmid pSMD1D3H is lacking the D2-domain and encodes a fusion protein containing the complete D1- and D3-domains with a carboxyl-terminal His tag. Plasmid pSMD3H lacks the D1- and D2-domains and encodes the entire D3-domain with a carboxyl-terminal His tag. Each plasmid was transfected into COS-7 cells, and the proteins expressed were isolated from the medium and analyzed under reducing conditions as described for Fig. 2. Fig.3 shows that pSMD1D2H (lane 2) and pSMD1D3H (lane 3) expressed M r∼ 180,000 and 170,000 proteins, respectively, that were poorly secreted into the medium. In contrast, plasmid pSMD3H (lane 4) expressed the D3-domain (M r ∼ 90,000) in amounts similar to those expressed by plasmid pSMNH (lane 1), which encodes the entire amino-terminal region (Fig. 1). Fig. 3 also shows that without prior reduction, plasmid pSMD1D2H (lane 6) secreted, into the medium, monomers in very small amounts and mainly disulfide-linked oligomers that ran slightly faster than high molecular weight aggregates seen at the interface between the stacking and running gels. Plasmid pSMD1D3H (lane 7) expressed disulfide-linked oligomers and aggregates, but not monomers. Plasmid pSMD3H (lane 8) expressed primarily a protein with a size (M r ∼ 240,000) similar to that of myosin (M r ∼ 220,000), consistent with the formation of disulfide-linked trimers along with significant amounts of monomers (M r ∼ 80,000) and very small amounts of disulfide-linked aggregates of the D3-domain. As expected, unmutated plasmid pSMNH (lane 5) expressed monomers and trimers, although the latter barely entered the running gel that was intentionally made with a higher percentage of acrylamide than the gel shown in Fig. 2. Earlier studies (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) showed that interchain disulfide bond formation by the D-domains occurs in acidic compartments of the Golgi complex because monensin and other compounds that increase the pH of these compartments inhibit interchain disulfide bond formation. Fig.4 shows the effects of monensin on the proteins expressed by pSMNH and pSMD3H. Confirming earlier studies (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), plasmid pSMNH secreted monomers under reducing conditions (lane 1) and monomers and disulfide-linked trimers under nonreducing conditions (lane 5). However, as also expected, in the presence of monensin, only monomers were observed under reducing (lane 2) and nonreducing (lane 6) conditions. In contrast, formation of trimers of the D3-domain by plasmid pSMD3H was unaffected by monensin (lane 8), although no interfacial aggregates were observed. The D3-domain secreted in the presence of monensin migrated slightly faster than that in its absence (lane 7), suggesting that this domain is N-glycosylated and that processing of N-linked oligosaccharides is impaired just as for the entire amino-terminal region (21Perez-Vilar J. Eckhardt A.E. DeLuca A. Hill R.L. J. Biol. Chem. 1998; 273: 14442-14449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). On reducing gels, monomers of the D3-domain were observed in