The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

Abstract

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc beta(1-N)Asn with alkali, alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field 1H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing the N-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of approximately 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing the N-linked carbohydrates as oligosaccharide-alditols.

Reductive Alkaline Release of N‐Glycans Generates a Variety of Unexpected, Useful Products

“…As the release conditions originally supposed by Carlson were optimized for O‐glycans, we followed the example of others and varied the conditions, albeit in smaller steps. These experiments were done with the A 4 A 4 ‐GENR glycopeptide, basic conditions for cleanup and basic solvent for PGC‐LC‐ESI‐MS throughout.…”

“…As several references point at a low yield upon alkaline release under Carlson conditions, we compared the peak area of the fibrin glycan obtained by chemical versus enzymatic release with LC‐ESI‐MS. Surprisingly, the area of the enzymatic glycan peaks was smaller, presumably because the pronase glycopeptides in part contained terminal Asn‐residues.…”

“…No more than 20% of fetuin N‐glycans was liberated under these conditions but apparently with the sialic acids remaining on the glycans . An only partial release was confirmed later in a study that obtained 95% release of ribonuclease B N‐glycans using stronger alkali and higher temperature . Later on, unintended “bycatch” of N‐glycans by the Carlson conditions was repeatedly observed .…”

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

“…Itoh et al ( 1986) also have provided evidence that yeast-derived preS2+S protein contains both N-linked and O-linked glycans by removing carbohydrate using endo H digestion and mild alkali treatment, respectively. However, there is increasing evidence that some N-linked glycans can be released by mild alkali treatment (Argade et al, 1989;Debray et al, 1984); thus, there is no definitive evidence to date for O-glycosylation in preS2+S proteins. Our preS2+S is presumed to contain carbohydrate only on Asn 4 since amino acid sequencing from the N-terminus indicated that the fourth amino acid was blocked.…”