Neil Jentoft

Why are proteins O-glycosylated?

The O-linked oligosaccharides of glycoproteins are usually clustered within heavily glycosylated regions of the peptide chain. Steric interactions between carbohydrate and peptide within these clusters induce the peptide core to adopt a stiff and extended conformation and this conformational effect appears to represent a major function of O-glycosylation.

Analysis of sugars in glycoproteins by high-pressure liquid chromatography

A method for analyzing the carbohydrate composition of glycoproteins and similar glycoconjugates by methanolysis followed by reverse-phase high-pressure liquid chromatography of the perbenzoylated methyl glycosides has been developed. As described, the method is capable of quantifying sugars in the 1- to 10-nmol range while further optimization of procedures may increase the usable sensitivity by a factor of 10 or greater. Improved yields of the sugar derivatives have been achieved by incorporating several modifications of the original methanolysis procedure. This, together with the use of high-pressure liquid chromatography rather than gas chromatography for separating the sugar derivatives, eliminates the need for empirically determined molar response ratios.

Protein labeling by reductive methylation with sodium cyanoborohydride: Effect of cyanide and metal ions on the reaction

Abstract Previous studies ( N. Jentoft and D. G. Dearborn, 1979 , J. Biol. Chem.254, 4359) have demonstrated that reductive methylation with labeled formaldehyde and NaCNBH3 provides a simple method for specifically labeling the amino groups of proteins using extremely mild reaction conditions. However, cyanide, which is one of the products of the reaction, reduces labeling effciency by reacting with formaldehyde to from the formaldehyde cyanohydrin addition product. Certain transition metal ions are able to prevent this secondary reaction by forming stable coordination complexes with cyanide. Inclusion of millimolar quantities of Ni(II) in reaction mixtures leads to a 20–30% increase in protein labeling so that maximal derivatization of amino groups can be realized with only a 3- to 4-fold ratio of formaldehyde to amine rather than the 5- to 10-fold excess necessary in the absence of metal ions.

Light-scattering studies of fractionated ovine sub-maxillary mucins

Static and dynamic light-scattering studies of solutions of ovine submaxillary mucin (OSM) glycoproteins, fractionated by exclusion chromatography on Sephacryl S-1000, are reported. These experiments yielded information regarding the structure and conformation of the glycoprotein chain, in the form of weight-average molecular weights, Mw, z-average radius of gyration, Rg,z, and z-average of the inverse hydrodynamic radius, (Rh-1)z. The values of (Rh-1)z are found to correlate very well with the S-1000 elution volume characteristics for four OSM fractions of different molecular weights. The structural parameters for these OSM fractions are, within experimental error, similar to those deduced for porcine submaxillary mucins (PSM) in earlier studies. The results suggest that, like PSM, the glycoprotein structure of OSM consists of linear chains constructed by covalently linking two or more elementary subunits together via disulfide bonds. In addition, the rigidity of the protein core of OSM is substantially greater than that observed for non-glycosylated-polypeptide random coils. Because (Rh-1)z, and hence, elution volume depends only on the molecular weight of the mucin protein core, the Mw calibration obtained for OSM should be applicable to the chromatography of other mucin glycoproteins.

Glycosyltransferases and Glycoprotein Biosynthesis

Since the discovery that sialic acid was the receptor site for influenza virus on the red blood cell (Gottschalk, 1960), recognition of the biological importance of glycoproteins and glycolipids has been increasing. Oligosaccharide moieties of these complex carbohydrates function as the primary antigenic determinants of blood-group substances, and may be involved in such diverse biological phenomena as contact inhibition and cell-cell adhesion of cultured cells, gamete recognition, transplant rejection, and recognition of specific receptor sites for hormones, viruses, and agglutinins. In addition, a large and increasing number of pathological conditions have been shown to be related to complex carbohydrates, including the glycosphingolipid storage diseases (see Chapter 11), cholera (Holmgren et al., 1973), herpes (Nahmias and Roizman, 1973), neoplasia (Burger and Martin, 1972), diabetes (Spiro and Spiro, 1971) hepatic cirrhosis (Marshall et al., 1974), and hemostasis (Barber and Jamieson, 1971). A better understanding of the chemistry and the mechanisms of biosynthesis of these complex molecules may provide information leading to the control of these diseases.