Immunologically cross-reactive 57 kDa and 53 kDa glycoprotein antigens of bovine milk fat globule membrane: isoforms with different N-linked sugar chains and differential glycosylation at early stages of lactation

Abstract

Two glycoprotein antigens with molecular masses of 57 kDa (MGP57) and 53 kDa (MGP53) were co-purified from bovine milk fat globule membrane (MFGM) by immunoaffinity chromatography using a monoclonal antibody raised against the MFGM. Their N-terminal sequences of 22 amino acids determined were identical, and the sequence was homologous (about 60% identical) to the deduced amino acid sequence of mouse milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) (Ref. [12], Stubbs, J.D. et al., Proc. Natl. Acad. Sci. USA, 87, 8417–8421, 1990). This suggests that MGP57/53 are bovine MFGM components 15/16 (PAS-6 and PAS-7), which have recently been reported to be bovine homologs of MFG-E8. N-Glycanase treatment of these glycoproteins reduced their molecular masses, and consequently the enzymatically deglycosylated MGP57 and MGP53 converged on a single band of 50 kDa as measured by SDS-PAGE, indicating that the polypeptide portions of these two distinct glycoprotein antigens are very similar or identical and that their N-linked sugar chains contributed to minor difference in their molecular masses. Western blot analyses using lectins also revealed that they differentially glycosylated; MGP57 was stained with concanavalin A (Con A) more strongly than MGP53, whereas MGP 53 was stained well with soybean agglutinin (SBA). Reactivity with SBA remarkably increased during early stages of lactation. Two-dimensional gel electrophoresis showed that MGP57 and MGP53 were electrically heterogeneous; from day 9 after parturition, both glycoproteins fell in almost the same range of isoelectric points between 6.4 and 7.6, also, such glycoproteins from day 1 after parturition were more acidic, probably due to terminal sialylation of their sugar chains.