Primary structural requirements for <i>N</i>‐glycosylation of peptides in rat liver

Bause, Ernst; Hettkamp, Harald

doi:10.1016/0014-5793(79)80559-1

Cited by 121 publications

(91 citation statements)

References 11 publications

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“…The attachment of glycan to a particular asparaginyl residue is governed by protein sequence (5,6). It has been determined, from studies involving amino acid substitutions of the ϩ2 residue following the asparagine residue, that a proximal hydrogen-bond acceptor is necessary for rendering the asparagine residue sufficiently nucleophilic to displace the GlcNAc 2 -Man 9 Glc 3 from the dolichol donor (7,8).…”

Section: From the Department Of Process And Product Development Amgementioning

confidence: 99%

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

Valliere-Douglass

Kodama

Mujacic

et al. 2009

Journal of Biological Chemistry

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We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C H 1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the C H 2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN 162 SGAL in the C H 1 domain of the antibody. This highly atypical modification is present at levels of 0.5-2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the C H 1 domain sequence in a recombinanthuman IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the ؊1 and ؉1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.

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Section: From the Department Of Process And Product Development Amgementioning

confidence: 99%

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

Valliere-Douglass

Kodama

Mujacic

et al. 2009

Journal of Biological Chemistry

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“…A fixed modification was set for 57 Da on cysteine and a variable modification of 16 Da on methionine resulting from alkylation and reduction steps, respectively. A variable 1 Da modification was set for asparagine on PNGase F + trypsin digested samples to account for the conversion of asparagine to aspartic acid after cleavage of glycan chains with the use of PNGase F (Plummer et al 1984), which takes place specifically at the consensus sequence Asn-XxxSer/Thr where Xxx can be any amino acid except proline (Bause & Hettkamp 1979).…”

Section: Mass Spectrometry and Database Searchingmentioning

confidence: 99%

Protein recycling in Bering Sea algal incubations

Moore

Harvey

Faux

et al. 2014

Mar. Ecol. Prog. Ser.

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Protein present in phytoplankton represents a large fraction of the organic nitrogen and carbon transported from its synthesis in surface waters to marine sediments. Yet relatively little is known about the longevity of identifiable protein in situ, or the potential modifications to proteins that occur during bloom termination, protein recycling and degradation. To address this knowledge gap, diatom-dominated phytoplankton was collected during the Bering Sea spring blooms of 2009 and 2010, and incubated under darkness in separate shipboard degradation ex periments spanning 11 and 53 d, respectively. In each experiment, the protein distribution was monited over time using shotgun proteomics, along with total hydrolyzable amino acids (THAAs), total protein, particulate organic carbon (POC) and nitrogen (PN), and bacterial cell abundance. Identifiable proteins, total protein and THAAs were rapidly lost during the first 5 d of enclosure in darkness in both incubations. Thereafter the loss rate was slower, and it declined further after 22 d. The initial loss of identifiable biosynthetic, glycolysis, metabolism and translation proteins after 12 h may represent shutdown of cellular activity among algal cells. Additional peptides with glycan modifications were identified in early incubation time points, suggesting that such protein modifications could be used as a marker for internal recycling processes and possibly cell death. Protein recycling was not uniform, with a subset of algal proteins including fucoxanthin chlorophyll binding proteins and RuBisCO identified after 53 d of degradation. Non-metric multidimensional scaling was used to compare the incubations with previous environmental results. The results confirmed recent observations that some fraction of algal proteins can survive water column recycling and undergo transport to marine sediments, thus contributing organic nitrogen to the benthos.

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“…Since Asn218 is embedded in the tripeptide sequence Asn-Pro-Ser, this site is not used for carbohydrate substitution (Bause, 1979;Bause and Hettkamp, 1979;Pohl et al, 1984). A d 8 4 is only partially glycosylated leading to glycoprotein variants with two or three oligosaccharide chains.…”

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confidence: 99%

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain

Pfeiffer

Strube

Schmidt

et al. 1994

European Journal of Biochemistry

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Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-P-Nacetylglucosaminidase H or peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glycans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing.The results revealed that Asnll7 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eul: J. Biochem. 186,273-2861. In contrast, As11117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75 %) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Am67 (YN-tPA) or Am58 (GS-tPA) as well as those at Asn184 and Am448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gala3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats.Our study clearly demonstrates that creation of a new glycosylation site at As1158 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn1 17, whereas introduction of a new site at Am67 did not. The relative amounts of complex-type glycans at Asnll7 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at that position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.

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Primary structural requirements for N‐glycosylation of peptides in rat liver

Cited by 121 publications

References 11 publications

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

Protein recycling in Bering Sea algal incubations

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain

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