Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex: A new member of the epidermal growth factor superfamily

Zeqi Sheng, Kai Wu, Kermit L. Carraway, Nevis L. Fregien

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Abstract

Ascites sublines of the 13762 rat mammary adenocarcinoma have a cell surface sialomucin complex composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the membrane-associated glycoprotein ASGP-2. The sialomucin complex is synthesized as a high Mr precursor, pre-sialomucin complex (pSMC-1). To characterize the structure of the membrane-associated component of this complex, a λgt11 cDNA expression library was constructed using mRNA from 13762 rat mammary adenocarcinoma cells and screened with polyclonal antibody against ASGP-2. The strongest antibody-binding clone, designated λASGP2.9-1, had a 1.3-kilobase (kb) insert, and hybridized to a 9-kb transcript in 13762 cell mRNA. The large size of this transcript was expected, since the estimated molecular mass of pSMC-1 is >250 kDa. To obtain the full sequence of ASGP-2, a longer cDNA (5.4 kb), designated pASGP1/2.1, was subsequently cloned by screening a plasmid library with an oligonucleotide complementary to the 5′ end of the phage insert. The amino acid sequence derived from nucleotide sequence of pASGP1/2.1 showed a 12-amino acid identity with amino acid sequence obtained from the NH2 terminus of ASGP-2, indicating the entire ASGP-2 coding region was included in the cDNA. Furthermore, an 18-amino acid identity with the NH2 terminus of a 6-kDa CNBr fragment of ASGP-2 was also observed in the cDNA sequence. The polypeptide contains several distinct domains, including a hydrophobic transmembrane domain, a short (20 residue) COOH-terminal cytoplasmic tail, and a large extracellular domain with 24 potential N-glycosylation sites. These properties correspond to features of ASGP-2 and pSMC-1 predicted by previous biochemical studies. Most interestingly, the extracellular domain contains two cysteine-rich sequences, each of which has a segment with strong similarities to proteins with epidermal growth factor activity. Since our recent studies show that ASGP-2 can modulate epidermal growth factor receptor phosphorylation activity, these results provide structural evidence to support the role of the heterodimeric sialomucin complex as a bifunctional modulator of cellular interactions and cell proliferation.

Original languageEnglish
Pages (from-to)16341-16346
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number23
StatePublished - Aug 15 1992

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Mucin-4
Sialomucins
Cloning
Molecular Cloning
Epidermal Growth Factor
Adenocarcinoma
Breast
Complementary DNA
Amino Acids
Rats
Amino Acid Sequence
Cell Proliferation
Glycosylation
Membranes
Messenger RNA
Phosphorylation
Bacteriophages
Antibodies
Membrane Glycoproteins
Cell proliferation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex : A new member of the epidermal growth factor superfamily. / Sheng, Zeqi; Wu, Kai; Carraway, Kermit L.; Fregien, Nevis L.

In: Journal of Biological Chemistry, Vol. 267, No. 23, 15.08.1992, p. 16341-16346.

Research output: Contribution to journalArticle

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abstract = "Ascites sublines of the 13762 rat mammary adenocarcinoma have a cell surface sialomucin complex composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the membrane-associated glycoprotein ASGP-2. The sialomucin complex is synthesized as a high Mr precursor, pre-sialomucin complex (pSMC-1). To characterize the structure of the membrane-associated component of this complex, a λgt11 cDNA expression library was constructed using mRNA from 13762 rat mammary adenocarcinoma cells and screened with polyclonal antibody against ASGP-2. The strongest antibody-binding clone, designated λASGP2.9-1, had a 1.3-kilobase (kb) insert, and hybridized to a 9-kb transcript in 13762 cell mRNA. The large size of this transcript was expected, since the estimated molecular mass of pSMC-1 is >250 kDa. To obtain the full sequence of ASGP-2, a longer cDNA (5.4 kb), designated pASGP1/2.1, was subsequently cloned by screening a plasmid library with an oligonucleotide complementary to the 5′ end of the phage insert. The amino acid sequence derived from nucleotide sequence of pASGP1/2.1 showed a 12-amino acid identity with amino acid sequence obtained from the NH2 terminus of ASGP-2, indicating the entire ASGP-2 coding region was included in the cDNA. Furthermore, an 18-amino acid identity with the NH2 terminus of a 6-kDa CNBr fragment of ASGP-2 was also observed in the cDNA sequence. The polypeptide contains several distinct domains, including a hydrophobic transmembrane domain, a short (20 residue) COOH-terminal cytoplasmic tail, and a large extracellular domain with 24 potential N-glycosylation sites. These properties correspond to features of ASGP-2 and pSMC-1 predicted by previous biochemical studies. Most interestingly, the extracellular domain contains two cysteine-rich sequences, each of which has a segment with strong similarities to proteins with epidermal growth factor activity. Since our recent studies show that ASGP-2 can modulate epidermal growth factor receptor phosphorylation activity, these results provide structural evidence to support the role of the heterodimeric sialomucin complex as a bifunctional modulator of cellular interactions and cell proliferation.",
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