Effect of the carbohydrate moiety on the secondary structure of .beta.2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins

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Abstract
By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma .beta.2-glycoprotein I (.beta.2I) were modified by sequential enzymatic degradation. The released monosaccharies (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoproein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of .beta.2I possesses more bi- than tri-antennans, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native .beta.2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed .beta.2I and most of the derivatives to contain predominantly .beta.-sheet and .beta.-turn structures. The lack of .alpha.-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of .beta.2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of .beta.-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed .beta.-turns after removal of 96% of the carbohydrate moiety. In 2-CE the position of the wavelength minimum was unaltered after each enzymatic step; however, a large increase in the magnitude of the molar ellipticity was seen as well as a 10% increase in .alpha.-helix and a 5% increase in random coil with corresponding reductions in both .beta.-sheet and .beta.-turn. Analysis of the primary structure of .beta.2I by secondary structure prediction methods which were modified to include the elements of secondary structure estimated from the CD spectra showed the presence of 31 .beta.-turns and 13 regions of .beta.-sheet and showed the remainder to be random coil. These results suggest that the transfer of large-size Man-type (oligomannose) N-glycans during biosynthesis of gp may ensure the proper folding of their polypeptide chains and, thus, the presence of glycans may be a requirement for the biological activity of .beta.2I.