Cloning, structure and expression of cDNA for mouse contrapsin and a related protein.
A cDNA clone (lambda MC-2) for contrapsin, a serine-proteinase inhibitor, was isolated from a lambda ZAP mouse liver cDNA library. The 1.6 kb cDNA insert of lambda MC-2 contained an open reading frame that encodes a 418-residue polypeptide (46,970 Da), in which a signal peptide of 21 residues was identified by comparison with the N-terminal sequence of the purified protein. The predicted structure (MC-2) also contained other peptide sequences determined by Edman degradation. Four potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (63 kDa). Further screening of the cDNA library with an EcoRI-EcoRI fragment (510 bp) of lambda MC-2 as a probe yielded another cDNA clone (lambda MC-7), which encodes a 418-residue polypeptide (MC-7) with a calculated mass of 47,010 Da. MC-2 showed 83% similarity at the amino acid level to MC-7, in contrast with 44% similarity to alpha 1-proteinase inhibitor. The possible reactive site (P1-P'1) for serine proteinase is suggested to be Lys-Ala for MC-2 and Ser-Arg for MC-7. Northern-blot analysis revealed that both MC-2 and MC-7 mRNAs have the same size of 1.8 kb and are markedly induced in response to acute inflammation. Construction of the expression plasmids pSVMC-2 and pSVMC-7 and their transfection into COS-1 cells demonstrated that pSVMC-2 directs the synthesis of a 63 kDa form whereas pSVMC-7 expresses a 56 kDa form. The difference in molecular mass between the two may be explained by the fact that the MC-7 sequence contains three potential sites for N-glycosylation, one site less than that of MC-2.
Ohkubo
K
K
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Ogata
S
S
Misumi
Y
Y
Takami
N
N
Sinohara
H
H
Ikehara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D015843:Serpins; D014162:Transfection; D014361:Trypsin Inhibitors / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D015843:Serpins; D014361:Trypsin Inhibitors; C033691:contrapsin; D004247:DNA