Glycylhydroxyprolyl Sequences in Earthworm Cuticle Collagen: Glycylhydroxyprolylserine

Goldstein, A.; Adams, Elijah

doi:10.1016/s0021-9258(18)62778-4

Cited by 43 publications

(8 citation statements)

References 23 publications

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“…The amino acid composition was consistent with the published data. 22 Compared with the bovine collagens, the amounts of Hyp, Thr, and Ser were high, whereas Pro and hydroxylysine were present in extremely low amounts. By our novel method, a markedly high peak of APDS-Gly-4Hyp was detected for earthworm collagen (Figure 5).…”

Section: ■ Results and Discussionmentioning

confidence: 93%

“…21,22 This finding was obtained by N-terminal amino acid sequence analysis of collagenase-released Gly-Xaa-Yaa tripeptides, but the method could not confidently determine the positional distribution of 4Hyp due to carryover from preceding cycles. 21,22 We extracted collagen from earthworm cuticles using acetic acid and performed amino acid analysis (Table S3). The amino acid composition was consistent with the published data.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Acid-solubilized collagen was prepared from earthworm cuticles as described previously . In brief, cuticles were removed from earthworms obtained from a market after a freeze–thaw procedure.…”

Section: Methodsmentioning

confidence: 99%

“…Acid-solubilized collagen was prepared from earthworm cuticles as described previously. 22 In brief, cuticles were removed from earthworms obtained from a market after a freeze−thaw procedure. After extensively washing the cuticles with cold distilled water, collagen was extracted with 0.5 M acetic acid at 4 °C overnight.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…We validated the efficiency of peptide generation by partial acid hydrolysis using model peptides and optimized the LC conditions for separating APDS-derivatized dipeptides. We analyzed bovine fibrillar collagens, including type I, III, and V collagens, as well as earthworm cuticle collagen in which Pro residues at the Xaa position are reported to be substantially 4-hydroxylated. , The quantitative accuracy of our method was verified by Edman sequencing followed by LC–MS detection of released phenylthiohydantoin (PTH) derivatizes of Pro, 4Hyp, and 3Hyp.…”

mentioning

confidence: 99%

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Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC–MS with Partial Acid Hydrolysis and Precolumn Derivatization

2020

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Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC−MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl-Nhydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.

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Section: ■ Results and Discussionmentioning

confidence: 93%