O‑Glycosylation and Hydroxylation of Amino Acid Residues of Collagen α‑1 (II) chain from Bovine Cartilage
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ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Bos Taurus (ncbitaxon:9913)
SUBMITTER: Haixu Tang, Yehia Mechref
PROVIDER: MSV000079174 | MassIVE | Wed Jun 24 21:26:00 BST 2015
REPOSITORIES: MassIVE
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Project description:O-Glycosylation of collagen is a unique type of posttranslational modifications (PTMs) involving the attachment of galactose (Gal) or glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also, hydroxyproline (HyP) result from the posttranslational hydroxylation of some proline residues in collagen. Here, LC-MS/MS was effectively employed to identify 23 O-glycosylation sites and a large number of HyP residues associated with bovine type II collagen ?-1 chain (CO2A1). The modifications of the 23 O-glycosylation sites varied qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied 22 of the identified glycosylation sites, while K773 was observed as unmodified. A large number of HyP residues at Yaa positions of Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed. Notably, HyP residue of Gly-HyP-Gln motif was detected, which has not been previously reported. Moreover, the deamidation of 8 Asn residues was identified, of which 2 Asp residues were observed at different retention times because of isomerization (Asp vs isoAsp). Partial macroheterogeneities of some CO2A1 glycosylation sites were revealed by LC-MS/MS analysis. ETD experiments revealed partial macroheterogeneities associated with K299-K308, K452-K464, K464-K470, and K857-K884 glycosylation sites. Semiquantitative data suggest that the glycosylation of hydroxylysine residues is site-specific.
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Project description: Not available
2015-06-30 | MSV000079177 | MassIVE
Project description:The covalent structure of the first 111 residues from the N-terminus of peptide alpha1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552-661 of the alpha1(II) chain. The sequence was determined by automated Edman degradation of peptide alpha1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the alpha1(II) chain with the homologous segment of the alpha1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the alpha1(II) chain (Butler, Miller & Finch (1976) Biochemistry15, 3000-3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the alpha1(II) chain were compared with the homologous regions of the alpha1(I) and alpha2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the alpha1(I) chain, it appears that the alpha1(II)-chain sequence presented here contains three more amino acids than that reported for the alpha1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide alpha1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
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Project description:?1(V) is an extensively modified collagen chain important in disease.Comprehensive mapping of ?1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets.The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties.Positions, numbers, and occupancy of modified sites can provide insights into ?1(V) biological properties. Aberrant expression of the type V collagen ?1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the ?1(V) chain are linked to lung transplant rejection and atherosclerosis. The ?1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the ?1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental ?1(V) and human recombinant pro-?1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these ?1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed ?1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of ?1(V) chain and type V collagen properties.
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Project description:Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells were studied by using continuous labelling and pulse-chase labelling experiments with [14C]lysine. Control experiments with [14C]proline indicated that in continuous labelling the hydroxylation of [14C]proline became linear with time after about 4 min and the secretion of collagen after about 35 min, as reported previously. In similar experiments with [14C]lysine the hydroxylation of [14C]lysine and the glycosylations of hydroxy[14C]lysine became linear at about 4 min, suggesting that these reactions were initiated while the polypeptide chains were growing on the ribosomes. Pulse-chase labelling experiments with [14C]lysine indicated that after a 5 min pulse-label the hydroxylation of [14C]lysine and the glycosylations of hydroxyl[14C]lysine continued during the chase period for about 20 min. The data suggest that these reactions are continued after the release of complete polypeptide chains into the cisternae of the endoplasmic reticulum, whereas the reactions are probably not continued after the formation of the triple helix and the movement of the molecules into the Golgi vacuoles.
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Project description:Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms ? sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.
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Project description:Epitopes of phospholipase A2 receptor (PLA2R), the target antigen in idiopathic membranous nephropathy (iMN), must be presented by the HLA-encoded MHC class II molecules to stimulate autoantibody production. A genome-wide association study identified risk alleles at HLA and PLA2R loci, with the top variant rs2187668 within HLA-DQA1 showing a risk effect greater than that of the top variant rs4664308 within PLA2R1. How the HLA risk alleles affect epitope presentation by MHC class II molecules in iMN is unknown. Here, we genotyped 261 patients with iMN and 599 healthy controls at the HLA-DRB1, HLA-DQA1, HLA-DQB1, and HLA-DPB1 loci with four-digit resolution and extracted the encoded amino acid sequences from the IMGT/HLA database. We predicted T cell epitopes of PLA2R and constructed MHC-DR molecule-PLA2R peptide-T cell receptor structures using Modeler. We identified DRB1*1501 (odds ratio, 4.65; 95% confidence interval [95% CI], 3.39 to 6.41; P<0.001) and DRB1*0301 (odds ratio, 3.96; 95% CI, 2.61 to 6.05; P<0.001) as independent risk alleles for iMN and associated with circulating anti-PLA2R antibodies. Strong gene-gene interaction was noted between rs4664308(AA) and HLA-DRB1*1501/DRB1*0301. Amino acid positions 13 (P<0.001) and 71 (P<0.001) in the MHC-DRβ1 chain independently associated with iMN. Structural models showed that arginine13 and alanine71, encoded by DRB1*1501, and lysine71, encoded by DRB1*0301, facilitate interactions with T cell epitopes of PLA2R. In conclusion, we identified two risk alleles of HLA class II genes and three amino acid residues on positions 13 and 71 of the MHC-DRβ1 chain that may confer susceptibility to iMN by presenting T cell epitopes on PLA2R.
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