CHARACTERIZATION OF GLYCOPROTEIN PAS-6 7 FROM MEMBRANES OF BOVINE-MILK FAT GLOBULES/

Glycoprotein components PAS-6 and PAS-7 were purified from bovine milk -fat-globule membranes and the amino acid sequence of their common pol ypeptide core, PAS-6/7, was determined by peptide and cDNA sequencing. The cDNA encoded a signal peptide of 18 amino acid residues and a mat ure PAS-6/ 7 protein of 409 amino acid residues. A cDNA splice variant was identified by reverse transcription/ PCR. Results obtained by ami no acid analyses, amino-acid-sequence analyses, carbohydrate-compositi on determinations, and MS analyses of glycopeptides revealed that both proteins were glycosylated with a carbohydrate structure that contain ed galactose, N-acetylgalactosamine and fucose, and which was O-linked to Ser9 in PAS-6 and to Thr16 in PAS-7. In addition, PAS-6 and PAS-7 were N-glycosylated at Asn41 with a hybrid-type-carbohydrate structure . A high-mannose glycan was N-linked to Asn209 of PAS-6. The sequence of PAS-6/7 contained two epidermal growth factor (EGF)-like domains in the N-terminal region, the second of which contained an RGD cell-adhe sion sequence in an extended loop, The EGF-like domains were followed by a C-terminal tandem repeat, which showed 60-63% similarity to the C 1-C2 domain of blood-clotting factors V and VIII. The disulfide bonds within the C1-C2 domain were identified.