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Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact

Open AccessPublished:September 20, 2019DOI:https://doi.org/10.1074/jbc.L119.010468
      Eyre et al. (
      • Eyre D.R.
      • Weis M.
      • Rai J.
      Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ) recently claimed that the histidine-involved collagen cross-links, histidinohydroxylysinonorleucine (HHL)
      The abbreviations used are: HHL
      histidinohydroxylysinonorleucine
      HHMD
      dehydrohistidinohydroxymerodemosine.
      2The abbreviations used are: HHL
      histidinohydroxylysinonorleucine
      HHMD
      dehydrohistidinohydroxymerodemosine.
      (
      • Yamauchi M.
      • London R.E.
      • Guenat C.
      • Hashimoto F.
      • Mechanic G.L.
      Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen.
      ) and dehydrohistidinohydroxymerodesmosine (HHMD) (
      • Tanzer M.L.
      • Housley T.
      • Berube L.
      • Fairweather R.
      • Franzblau C.
      • Gallop P.M.
      Structure of two histidine-containing cross-links from collagen.
      ), are both laboratory artifacts. We have several concerns about this study.
      1) Failure to identify these cross-linked peptides does not prove their “nonexistence.” The approaches of Eyre et al. are very different from ours (
      • Mechanic G.L.
      • Katz E.P.
      • Henmi M.
      • Noyes C.
      • Yamauchi M.
      Locus of a histidine-based, stable trifunctional, helix to helix collagen cross-link: stereospecific collagen structure of Type I skin fibrils.
      ). We prepared the insoluble fraction of bovine dermis, solubilized by repeated denaturation-trypsin treatments, purified the HHL cross-linked peptides by a series of chromatography under dissociative conditions, and further truncated and characterized them. It is highly unlikely that the proposed weakly linked peptides (see Fig. 7) (
      • Eyre D.R.
      • Weis M.
      • Rai J.
      Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ) remained together during these processes.
      2) Eyre et al. provide no direct evidence showing that the proposed peptides generate HHL and HHMD upon acid hydrolysis. Small amounts of HHL and HHMD found in an acid hydrolysate of pooled HPLC fractions (see Fig. 8) (
      • Eyre D.R.
      • Weis M.
      • Rai J.
      Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ), a mixture of peptides, do not identify the peptides of their origin.
      3) Their proposed peptide/cross-link structures are mostly deduced from the MS analysis. While MS is a powerful analytical tool, the data interpretation becomes challenging when the peptide is complex and large. Indeed, their MS data determine only the partial structure of the proposed peptides (see Figs. 6 and 7) (
      • Eyre D.R.
      • Weis M.
      • Rai J.
      Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ) and do not seem to correspond to the theoretical values. In addition, their proposed peptide (see Fig. 6) (
      • Eyre D.R.
      • Weis M.
      • Rai J.
      Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ) shows incomplete cleavages by collagenase, like CNBr digestion, implying there can be peptide variants that may have been missed.
      Thus, while the proposed ideas are interesting, these data alone are insufficient to support their claims.

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