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

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 monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (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-antennas, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of human recombinant β2‐glycoprotein I with anticardiolipin antibody cofactor activity

“…They appear to be glycoforms as digestion with peptide:N-glycosidase F which cleaves all N-linked oligosaccharides from asparagine residues resulted in a single diffuse band of 3642 kDa, the main portion of which co-migrates with similarly digested native fl2-glycoprotein I (40 kDa). This size coincides with the deglycosylation study of Walsh et al [18]. In the modified IgG cardiolipin-ELISA, anti-cardiolipin antibodies from five autoimmune patients purified using affinity and cation exchange chromatography [14] had an absolute requirement for recombinant fl2-glycoprotein I in order to bind cardiolipin (Fig.…”

Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.

“…CD spectra of apoB-17 on DMPC/apoB-17 complexes (protein concentration, .ug/ml) in 5 mM phosphate buffer (pH 7.4) were recorded in 0.2-or 0.5-mm cells from 250 to 200 nm on an AVIV 60 DS spectropolarimeter (AVIV Associates, Lakewood, NJ) at 25°C as described (23,24). Molar ellipticity values were calculated as described (24).…”

Phospholipid-Bound β2-Glycoprotein I Induces the Production of Anti-Phospholipid Antibodies

“…An exact interpretation of the spectral change is difficult, as the CD spectrum of 2GPI alone is atypical for a globular protein [19]. Previous studies have suggested that native 2GPI is 40% -sheet, 30% -turn, and 30% random coil, with no contribution from -helices [20]. Regardless of this, interactions between CL N and 2GPI resulted in a clear change in the CD spectrum, which indicates a change in the secondary structure of this protein.…”