Robert E. Hardy

Specific 13C reductive methylation of glycophorin A. Possible relation of the N-terminal amino acid and the lysine residues to MN blood group specificities

Heterozygous and homozygous glycophorin A were partially and fully reductively methylated with 13C-enriched formaldehyde in the presence of sodium cyanoborohydride. Total reductive methylation modified the five lysine residues (to produce N epsilon,N-[13C]dimethyl lysine) and the N-terminal amino acid residues (N alpha,N-[13C]dimethyl serine and leucine) of glycophorins AM and AN, respectively. 13C-NMR spectra of these species indicated that the 13C-enriched methyl carbons of the five lysyl derivatives all occur at 44.1 ppm downfield from Me4Si. Titration results indicate that the pK alpha of these methylated lysines is greater than 10. The chemical shift equivalent methyl resonances of the 13C-enriched methylated N-terminal Leu derivative were found to occur at 42.8 ppm downfield from Me4Si and exhibited a normal pH titration behavior (pK alpha approximately 7.4). The methyl resonances of the N alpha,N-[13C]dimethyl Ser derivative, on the other hand, were found to exhibit chemical shift nonequivalence, indicating rotational constraints about the C alpha-N bond. The linewidths of the two methyl resonances were also found to be considerably different; this phenomenon could be eliminated by running spectra of the sample (pH approximately 5.0) at elevated temperatures (75 degrees C). This result suggested that for the N alpha,N-[13C]dimethyl Ser derivative of glycophorin AM, hindered rotation must occur about one of the N alpha-13CH3 bonds. This structural difference at the N-terminal residue of glycophorins AM and AN may be related to the MN blood group determinants displayed by these related glycoproteins.

Possible role of the carbohydrate residues in the display of the MN blood group determinants by glycophorina

Heterozygous glycophorin AM,N and homozygous glycophorin AM were reductively methylated with 13C-enriched formaldehyde in the presence of cyanoborohydride. Total reductive methylation modified the five lysine residues, and the N-terminal amino acid residues (serine and leucine) of glycophorins AM and AN, respectively. The 13C resonances of the incorporated labels were monitored as a function of the degree of glycosylation of the glycoprotein. While minimal, if any, structural changes were observed near the N-terminal amino acid upon removal of alpha-D-N-acetylneuraminic acid residues, gross structural changes were observed when most of the oligosaccharide chains were removed. We also found that progressive methylation of the lysine residues of glycophorin AM may influence either the chemical shift of one of the nonequivalent methyl groups of the N alpha, N-[13C]dimethyl serine residue, or one of the two states of glycophorin AM.

Magnetic resonance study of glycophorin A-containing 13C-enriched methionines

Methionine‐81 and/or ‐8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with 13CH3I to produce the sulfonium ion derivatives [S‐[13C]methylmethionine‐8]glycophorin A and [S‐[13C]methylmethionine‐8 and ‐81]glycophorin A. 13C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me4Si. A spin‐lattice relaxation time of 0.4 was observed for the 13C‐enriched methyl resonances of the sulfonium ion derivatives of Met‐8 and ‐81, which corresponds to an effective correlation time of < 2× 10−10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2‐mercaptoethanol produces native glycophorin A which now has the ε‐carbon of the methionine residue(s) 45% isotopically enriched. The ε‐carbon of Met‐8 was found to occur at 15.7 ppm downfield from Me4Si whereas the ε‐carbon of Met‐81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me4Si. The spin‐lattice relaxation time of both resonances was found to be ∼0.3 s.

13C-N.M.R.-spectral study of some mono- and di-O-galactosylated dipeptides. Possible structural perturbations due to O-glycosylation

Abstract Carbon-13 nuclear magnetic resonance data for mono- and di- O -α- and -β- D -galactosylated dipeptides composed of Thr and Gly are presented. The results conclusively show that peptide-bond formation does not affect the chemical shifts of the attached carbohydrate carbon atoms. In the case of the di- O -glycosylated threonyl-threonine, no carbohydrate-carbohydrate interactions could be observed. For some of the mono- O -glycosylated dipeptides, the attached glycosyl group appears to have a peculiar effect on the chemical shifts of some of the carbon resonances of the amino acids.

13C-n.m.r. spectral study of 13C reductively methylated glycopeptides derived from glycophorin A

Abstract 13C-n.m.r. spectral data for 13C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the 13C-n.m.r. spectral data for the 13C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins AM and AN at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin AM.

Structural studies of the M blood group determinant

Structural studies of homozygous glycophorin AM were undertaken by monitoring the 13C methyl resonances of 13C reductively methylated glycophorin AM (contains five Nϵ,N-[13C]dimethyl Lys residues, and the N-terminal Nα,N-[13C]dimethyl Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin AM and deglycosylated glycophorin AM indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated AM.

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

Abstract 13C nuclear magnetic resonance (n.m.r.) spectral data for 13C reductively methylated N-terminal tryptic glycopeptides and for 13C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins AM and AN are presented. Their 13C chemical shift data are compared with the previously published 13C n.m.r. data for 13C reductively methylated homozygous glycophorins AM and AN in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the 13C resonances of N α ,N-[ 13 C ] dimethyl leucine of glyco-octapeptide AN and of N α ,N-[ 13 C ] dimethyl serine of glyco-octapepti AM indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of 13C reductively methylated glycophorin AM is lost when the glyco-octapeptide AM is produced. The 13C resonance of N α ,N-[ 13 C ] dimethyl leucine of glycooctapeptide AN titrated with a p K a of 7.7 (Hill coefficient ∼ 1 ). The 13C resonance of N α ,N-[ 13 C ] dimethyl serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for 13C-labelled glycooctapeptide AM molecule, the pH dependence of the chemical shift of the 13C resonance of N α ,N-[ 13 C ] dimethyl serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.

13C-N.M.R.-spectral study of the pH behavior of reductively [13C]methylated, glycophorin a glyco-octapeptides and a related glycopentapeptide

Abstract The pH dependence of the labeled-carbon resonances of reductively [ 13 C] methylated compounds tri- l -Ser, glyco-octapeptide A M , asialoglyco-octapeptide A M , glyco-octapeptide A N , asialoglyco-octapeptide A N , and a glycopentapeptide was investigated. The results are discussed relative to those previously observed for reductively [ 13 C]methylated, intact glycophorins A M and A N , and in terms of the mode of display of the MN blood-group specificities by these related glycoproteins. The results indicated that the α- d -NeuAc groups appear to affect the pH-titration results of glyco-octapeptides A M and A N . Moreover, comparison of the pH-titration results for reductively [ 13 C]methylated glyco-octapeptide A M and reductively [ 13 C]methylated asialoglyco-octapeptide A M with those of a reductively [ 13 C]methylated glycopentapeptide and reductively [ 13 C]methylated tri- l -Ser indicated that the other carbohydrate residues present (α- d -GalNAc and β- d -Gal) may also affect the pH-titration results. The reductive-methylation modification appears to affect the chemical shifts of the carbohydrate and peptide carbon atoms of the glycopentapeptide minimally.

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The 13C resonances of Nα,N-[13C]dimethylserine of partially 13C reductively methylated glycophorin AM were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the 13C resonances of Nα,N- [13C]dimethylserine of fully 13C reductively methylated deglycosylated glycophorin AM. The ‘major’ component of Nα,N- [13C]dimethylserine of glycophorin AM did not titrate, whereas the ‘minor’ component titrated with a pKa of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the Nα,N- [13C]dimethylserine resonances of 13C reductively methylated deglycosylated glycophorin AM.

13C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

13C nuclear magnetic resonance spectroscopy (13C n.m.r.) was used to determine the pH titration parameters for the N-terminal Nα,N-[13C]dimethylamino and Nα,N-[13C]monomethylamino groups of glycophorins AM and AN, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin AM.