Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312.

Sheares, B T

doi:10.1016/s0021-9258(18)37821-9

Cited by 13 publications

(4 citation statements)

References 24 publications

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“…The protein moiety is composed of 385 amino acids with a molecular mass of 43 kDa (Chart ). Ovalbumin has a single glycosylation site at 292 Asn with a second potential glycosylation at 311 Asn. − Figure shows the mass spectrum of glycopeptides obtained after Pronase digestion and PGC separation. Doping the sample with NaCl enhanced the intensities of the quasimolecular ion ([M + Na] + ) while the addition of KCl shifted the signals accordingly.…”

Section: Resultsmentioning

confidence: 99%

Determination of N-Glycosylation Sites and Site Heterogeneity in Glycoproteins

et al. 2003

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An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.

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Section: Resultsmentioning

confidence: 99%

Determination of N-Glycosylation Sites and Site Heterogeneity in Glycoproteins

et al. 2003

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“…To further explore the efficacy of bead immobilized pronase for site specific glycoprofiling, several proteins with increasingly complex glycosylation were subjected to pronase bead digestion and direct nESI-FTICR-MS analysis. These included the following: CEA, a 42.9 kDa glycoprotein with two potential N-glycosylation sites and a known population of high mannose and hybrid type N-glycans; [59][60][61] HAT, a 77.1 kDa glycoprotein having two consensus N-glycosylation sequons and carrying sialylated complex type N-linked oligosaccharides; [62][63][64][65] and BF, a 38.4 kDa glycoprotein containing three potential sites of N-glycan attachment, an array of sialylated complex type N-glycans, and several known and suspected sites of core 1 and core 2 type O-glycan modification. [66][67][68][69] The pronase bead digests of these glycoproteins are presented in Figure 3.…”

Section: Resultsmentioning

confidence: 99%

Analytical Performance of Immobilized Pronase for Glycopeptide Footprinting and Implications for Surpassing Reductionist Glycoproteomics

et al. 2008

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A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. In order to meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.

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“…The prospective sites for glycosylation in human cystatin C include amino acid residues at the positions 35, 36, and 79 (15). Since there were some reports in that the efficiency of the glycosylation decreases with the distance of the glycosylation signal from the N-terminus (14,(19)(20)(21), we chose the glycosylation site at the position 35 in the molecule. Thus, a mutant human cystatin C, A37S, in which the alanine residue was replaced by serine residue at the position 37 in order to obtain Asn 35 -Lys 36 -Ser 37 sequence, was constructed.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Anti-Rotavirus Action of Human Cystatin C by Site-Specific Glycosylation in Yeast

et al. 2004

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The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.

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Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312.

Cited by 13 publications

References 24 publications

Determination of N-Glycosylation Sites and Site Heterogeneity in Glycoproteins

Determination of N-Glycosylation Sites and Site Heterogeneity in Glycoproteins

Analytical Performance of Immobilized Pronase for Glycopeptide Footprinting and Implications for Surpassing Reductionist Glycoproteomics

Enhanced Anti-Rotavirus Action of Human Cystatin C by Site-Specific Glycosylation in Yeast

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