Treatment of human milk fat globules with pronase gave a soluble sialoglycopeptide mixture which was fractionated on Sephadex G-50 to give two major hexose-sialic acid peaks I and II. Material corresponding to peak II was treated with alkaline borohydride when an oligosaccharide component was released. Purification and gas chromatographic analysis of the released carbohydrate showed it to contain sialic acid; galactose, N-acetylglucosamine and N-acetylgalactosamine in the ratio 3:3.5:2:1. Sialoglycopeptides I and II both showed epithelial membrane antigenic activity in the radioimmunoassay of Ormerod and Steele. Further fractionation of peaks I and II by ion-exchange led, in the case of peak II, to a sialic acid-rich glycopeptide with increased antigenic activity. Papain digestion of human milk fat globule membrane gave a crude sialoglycopeptide mixture which also showed strong epithelial membrane antigenic activity. An investigation was made of means for selectively removing carbohydrate from glycopeptides. Hydrogen fluoride in pyridine was found to remove 67% of the total carbohydrate from fetuin at 45°C but very little at 0°C. In order to assess the effect of hydrogen fluoride and other deglycosylating agents on the structure and conformation of a polypeptide chain, a radioimmunoassay was developed for the antigenicity of IgG. Treatment of IgG with hydrogen fluoride at 45°C destroyed the antigenicity whereas treatment of IgG with a mixture of glycosidases from Trichomonas foetus caused no loss of antigenicity indicating that the glycosidases but not hydrogen fluoride can remove carbohydrate without affecting the polypeptide chain. Sialoglycopeptide II was treated with a mixture of exo- and endo-glycosidases from Diplococcus pneumoniae which removed 60% of the carbohydrate and led to a five-fold loss in epithelial membrane antigenic activity. Removal of sialic acid alone caused little affect indicating that the antigenic activity of Sialoglycopeptide II is carried by carbohydrate residues other than sialic acid.
|Date of Award||1981|