Isolation and Characterization of Mucin-Like Glycoprotein in Human Milk Fat Globule Membrane

Abstract

Milk fat globule membrane (MFGM) enclosing fat droplets in human milk was found to contain a high molecular weight glycoprotein which did not migrate in 10% acrylamide gel on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein (termed PAS-0) was isolated by Sepharose CL-4 B chromatography. Isolated PAS-0 gave one band on SDS-PAGE using 5% acrylamide gel (acrylamide : bisacrylamide=4:1, w/w) and gave one peak on analytical ultracentrifugation, indicating its homogeneity.
PAS-0 was rich in serine, threonine, proline, glycine, and alanine. In contrast, contents of sulfur-containing amino acids were very low. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were detected as constituent sugars of PAS-0 and the total carbohydrate content was about 50%. Alkaliborohydride treatment suggested that the carbohydrate moiety was linked to the polypeptide core with O-glycosidic bond (s). These results suggested that PAS-0 was a mucin-like glycoprotein. PAS-0 was shown to be resistant to pepsin, trypsin and chymotrypsin digestion, but susceptible to Pronase and Subtilisin BPN'. Extraction of intact milk fat globules (cream) with MgCl₂ and guanidine hydrochloride solutions suggested that PAS-0 was an intrinsic component of MFGM. Digestion of cream with Subtilisin BPN' demonstrated that PAS-0 was located on the external surface of fat globules and was accessible to molecules outside the globules. By agglutination-inhibition tests using eight lectins, PAS-0 was suggested to act as surface receptors for Ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin.