Glycopeptide Profiling Research Articles

Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.

The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.

Fucosyl Glycopeptide Profiles of Keratinocytes from Various Epithelial Tissues of the Rabbit in Relation to Differentiation In Vivo and In Vitro

Keratinocytes have been isolated from rabbit cornea, esophagus, and skin by trypsinization. The freshly isolated cells differed in their morphology, growth characteristics in culture, transglutaminase activity (a marker for differentiation), and the composition of glycoprotein-derived fucopeptides. A clear relationship has been shown between the proportion of low-molecular-weight fucopeptides and the pattern of keratinization, being minimal in cornea and maximal in skin. Comparison with earlier literature suggests that this relationship may be a general feature of epithelial tissue. After several passages in culture, all differences between the three cell types disappeared. The unusual elution pattern of the fucopeptides on gel filtration suggests the possibility of a block at an early stage of glycoprotein synthesis under culture conditions.

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.

The influence of urea and cell-surface protein on the behavior of nontumorigenic and chemically transformed cells.

Alterations in cell-cell interactions induced by urea and urea-isolated cell-surface protein (CSP) were examined using the nontumorigenic mouse fibroblast 10T1/2 cell line and the malignantly transformed MCA daughter cell line as a model system. Both cell lines were exposed to urea and CSP, and contact inhibition was quantitated based on nuclear overlaps. The Abercrombie Overlap index was found to be dependent on cell density, and a new method of overlap analysis was developed based on the regression of the number of overlaps per microscope field on the number of cells per field. Urea had a differential effect on both protein and deoxyribonucleic acid synthesis and a differential effect on the overlap regression in the two cell lines. Urea increased the regression coefficient in the normal (10T1/2) cell line while decreasing it in the transformed (MCA) cell line to approximately equal levels of overlap. Added CSP had no effects on overlaps in the MCA line but increased overlaps in the 10T1/2 line to the level of the MCA control, suggesting that the CSP interactions were more specific than the urea interactions. Trypsin-derived cell-surface glycopeptide profiles of the two cell lines showed a difference consistent with previously reported differences between normal and transformed cell lines. However, the glycopeptide profile of the urea-isolated CSP from the normal 10T1/2 cell line and the trypsin-derived glycopeptides of the transformed MCA cell line were not significantly different in the high-molecular-weight region, suggesting that the relative abundance of CSP may be higher in the MCA line, and the fact that the addition of CSP to the 10T1/2 line increased overlap tendency to the level of the MCA line suggested that CSP is an important factor in the modulation of overlap tendency. Urea and CSP may exert their effects on overlap tendency by affecting the integrity or order of the cell-surface components. The increased overlap tendency of the 10T1/2 line in the presence of CSP was not associated with increased cell density. Density dependence of cell growth was apparently not directly related to density dependence of overlap tendency. Many xenobiotics have surface-active properties and are known to inhibit protein synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Separation of glycopeptides by high performance liquid chromatography.

A method is presented for separation of tryptic glycopeptides-containing oligosaccharides of the N-asparagine-linked type. High performance liquid chromatography (HPLC) of glycopeptides on a C18 reverse-phase system eluted with a gradient of 0%-50% acetonitrile in 0.1 M NaPO4 pH 2.2 resolves the two major glycosylation sites from the envelope glycoprotein (G) of vesicular stomatitis virus. Glycopeptides containing N-linked oligosaccharides of the complex type coelute with those containing N-linked oligosaccharides of the neutral, high mannose type, indicating that separation is based upon peptide rather than carbohydrate composition. The contribution of the carbohydrate component to glycopeptide elution, as determined by cleavage of the high mannose oligosaccharides with endo-beta-Nacetylglucosaminidase H, is that of a significant, but minor, decrease in peptide retention time. Comparison of the tryptic glycopeptide profiles of G isolated from both wild type and mutant strains of VSV illustrates the rapid, reproducible, and quantitative nature of the technique. Through HPLC analysis of appropriately treated glycopeptides, it is possible to explore both the nature and extent of glycosylation at individual sites in glycoproteins in a single step.

Relationship of GIX antigen expression to the glycosylation of murine leukemia virus glycoprotein.

The GIX antigen, which is expressed on the surface of thymocytes of certain mouse strains, is an antigenic determinant of the major envelope glycoprotein of murine leukemia virus (gp70). Although GIX is expressed in some mouse strains that appear to be free of virus, the antigen can also be induced in GIX- mice by infection with particular murine leukemia viruses (termed GIX+). We have investigated the envelope gene products from two closely related viruses that differ in their GIX phenotype. Analysis of the envelope protein precursors by polyacrylamide gel electrophoresis and endoglycosidase treatment indicated that the GIX+ viral protein contained six oligosaccharide chains, whereas the GIX- viral protein contained seven. The observed differences in gel electrophoretic mobilities and glycopeptide profiles of the respective glycosylated envelope gene cleavage products (gp70) may be accounted for by the presence of an additional oligosaccharide chain on the gp70 of the GIX- virus. No differences between the apparent molecular weights of the nonglycosylated product of the envelope gene (p15E) were detected. These results suggest that the GIX- virus codes for an extra glycosylation site relative to the GIX+ virus, and this oligosaccharide chain is present both on the envelope gene precursor (Prenv) and on the major cleavage product (gp70). Recent nucleotide sequence analyses of selected RNase T1 oligonucleotides from the genomes of viruses that differ in GIX phenotype have similarly suggested that there may be a correlation between the GIX- phenotype and an extra glycosylation site [Donis-Keller, H., Rommelaere, J., Ellis, R. W. & Hopkins, N. (1980) Proc. Natl. Acad. Sci. USA 77, 1642-1645]. The results of these two different approaches raise the possibility that the presence of an additional oligosaccharide chain on gp70 may, either directly or indirectly, mask the expression of the GIX antigen on the surfaces of thymocytes and virus-infected cells.