Structural and Conformational Analysis of Glycan Moieties in Situ on Isotopically 13C,15N-Enriched Recombinant Human Chorionic Gonadotropin

Ct, Weller; Lustbader, Joyce W.; Seshadri, K.; Jm, Brown; Ca, Chadwick; Ce, Kolthoff; Ramnarain, Shakuntala; Pollak, Susan; Canfield, Robert E.; Sw, Homans

doi:10.1021/bi960432f

Cited by 66 publications

(43 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Radioisotope labeling ( 3 H or 14 C) of glycoprotein glycans has been reported to facilitate detection of small amounts of glycoprotein samples (47). Furthermore, stable isotope labeling of glycoproteins by metabolic and chemo-enzymatic methods has been reported for NMR analyses (48)(49)(50)(51)(52). However, for NMR analysis, it is necessary to establish a systematic method for labeling high molecular weight glycoproteins, such as IgG, with stable isotopes.…”

Section: B Establishment Of Stable Isotope Labeling Of Glycoproteinsmentioning

confidence: 99%

Untitled

Yamaguchi¹

2008

TIGG

View full text Add to dashboard Cite

Section: B Establishment Of Stable Isotope Labeling Of Glycoproteinsmentioning

confidence: 99%

Untitled

Yamaguchi¹

2008

TIGG

View full text Add to dashboard Cite

“…We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials. Given this access, we probed the effects of clustered glycosylation patterns on peptide conformation and recognition (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We have conducted extensive NMR and restrained molecular dynamics calculations (19,20) on fully synthetic clustered carbohydrate tumor antigens (1)(2)(3)(4) corresponding to a fragment of CD43, a glycoprotein aberrantly expressed on the surface of acute myelogenous leukemia cells (21)(22)(23)(24).…”

mentioning

confidence: 99%

Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins

Live

Williams

Kuduk

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cell-surface mucin glycoproteins are altered with the onset of oncogenesis. Knowledge of mucin structure could be used in vaccine strategies that target tumorassociated mucin motifs. Thus far, however, mucins have resisted detailed molecular analysis. Reported herein is the solution conformation of a highly complex segment of the mucin CD43. The elongated secondary structure of the isolated mucin strand approaches the stability of motifs found in folded proteins. The features required for the mucin motif to emerge are also described. Immunocharacterization of related constructs strongly suggests that the observed epitopes represent distinguishing features of tumor cell-surface architecture.The exterior of most cells is dominated by glycolipids, proteoglycans, and glycoproteins, including mucin-like proteins. These display polyvalent ␣-O-linked carbohydrates on proximal serine and threonine residues (1-4). Altered expression of cell-surface mucin character is often characteristic of malignant cells (1,3,5). Accordingly, tumor-associated mucins are good targets for a vaccination strategy (3, 5-9). We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials. Given this access, we probed the effects of clustered glycosylation patterns on peptide conformation and recognition (9-18). We have conducted extensive NMR and restrained molecular dynamics calculations (19, 20) on fully synthetic clustered carbohydrate tumor antigens (1-4) corresponding to a fragment of CD43, a glycoprotein aberrantly expressed on the surface of acute myelogenous leukemia cells (21-24). Our findings demonstrate how clustered glycosylation induces the peptide backbone into an unprecedented rigid scaffold corresponding to a polypeptide secondary conformation, which is consistent with elongated mucin glycoprotein structure and function (1,(25)(26)(27)(28)(29). Remarkably, the glycosylation-induced structure approaches the stability of motifs found in globular proteins.The CD43, or leukosialin, protein presented an ideal candidate from which to select a substructure for synthesis. In consequence of its possible role in inducing immunologic response, the system is relatively well characterized (22-24). The sequence STTAV is a glycosylation locus found in the amino terminus of the protein. We have accessed through chemical synthesis the clustered 2,6-STF trisaccharide 1 (7), which is present on CD43 when expressed on acute myelogenous leukemia cells (24). In addition, we have synthesized the TF disaccharide 2 and the monosaccharide Tn antigen 3. Through our synthetic methods, we also gained access to the ␤-linked stereoisomer of the TF antigen 4 (Fig.

show abstract

“…Chemically deglycosylated-oFSH and dg-hCG were reportedly more stable to heat denaturation at 100 C than their intact counterparts (Sairam & Manjunath 1982a, Sairam 1983, while Asn 52 hCG-glycosylation mutants were reportedly unstable at lower temperatures (Matzuk et al 1989, Heikoop et al 1998. The crystal structure of dg-urinary hCG suggested hydrogen bonds between the GlcNAc( 1-4)GlcNAc disaccharide remnant and hCG side chains (Lapthorn et al 1994); however, a nuclear magnetic resonance spectroscopy (NMR) study of intact recombinant hCG provided no evidence for hydrogen bonds between the biantennary oligosaccharide attached to Asn 52 and the hCG subunit (Weller et al 1996). The crystal structure of recombinant hFSH, bearing intact, high mannose oligosaccharides also indicated hydrogen bonds between hFSH and the Asn 52 oligosaccharide (Fox et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Truncated equine LH beta and asparagine(56)-deglycosylated equine LH alpha combine to produce a potent FSH antagonist

Butnev

Singh²,

Nguyen³

et al. 2002

Journal of Endocrinology

View full text Add to dashboard Cite

Hybrid hormone preparations were prepared by combining intact and Asn dg-eLH :eLH t for 72 h at 37 C resulted in no loss of FSH receptor-binding activity. Preincubation resulted in 50% loss of receptor-binding activity by the eFSH preparation due to subunit dissociation, while 88% of N 56 dg-eLH :eFSH activity was lost following 72 h, 37 C preincubation. While Asn 56 oligosaccharide had no effect on eLH hybrid stability, it did contribute to the stability of the eFSH heterodimer.

show abstract

Structural and Conformational Analysis of Glycan Moieties in Situ on Isotopically ¹³C,¹⁵N-Enriched Recombinant Human Chorionic Gonadotropin

Cited by 66 publications

References 23 publications

Untitled

Untitled

Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins

Truncated equine LH beta and asparagine(56)-deglycosylated equine LH alpha combine to produce a potent FSH antagonist

Contact Info

Product

Resources

About