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 polypeptide core, PAS‐6/7, was determined by peptide and cDNA sequencing. The cDNA encoded a signal peptide of 18 amino acid residues and a mature PAS‐6/7 protein of 409 amino acid residues. A cDNA splice variant was identified by reverse transcription/PCR. Results obtained by amino acid analyses, amino‐acid‐sequence analyses, carbohydrate‐composition determinations, and MS analyses of glycopeptides revealed that both proteins were glycosylated with a carbohydrate structure that contained 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 Am41 with a hybrid‐type‐carbohydrate structure. A high‐mannose glycan was N‐linked to Asn209 of PAS‐6. The sequence of PAS‐617 contained two epidermal growth factor (EGF)‐like domains in the N‐terminal region, the second of which contained an RGD cell‐adhesion sequence in an extended loop. The EGF‐like domains were followed by a C‐terminal tandem repeat, which showed 60–63% similarity to the C1‐C2 domain of blood‐clotting factors V and VIII. The disulfide bonds within the C1‐C2 domain were identified.