Advertisement

Reply to Yamauchi et al.: 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.RL119.010692
      Yamauchi et al. (
      • Yamauchi M.
      • Taga Y.
      • Terajima M.
      Letter to the Editor: Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
      ) question the data in our recent paper (
      • 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.
      ) as support for our conclusions. We disagree and maintain that histidinohydroxylysinonorleucine (HHL)
      The abbreviations used are: HHL
      histidinohydroxylysinonorleucine
      HHMD
      histidinohydroxymerodemosine.
      (
      • 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.
      ,
      • 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.
      ) does not exist as a natural product in collagen.
      In Point 1, “these cross-linked peptides” presumably refers to peptides that Mechanic et al. (
      • 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.
      ) thought were linked by HHL based on Edman N-terminal sequencing and amino acid analysis. In our opinion, this did not rule out a mixture of physically associated peptides or a peptide remnant of the C-telopeptide aldol adduct that we conclude creates HHL on acid hydrolysis (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.
      ). Quantifying HHL in acid hydrolysates of equal amounts of starting tissue collagen, a proteolytic digest of it, and subsequent fractions is highly instructive. For example, from bovine cornea and dermis, the yields of HHL in a bacterial collagenase digest are 6 and 17% based on the LC/MS assay method used in 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.
      ).
      D. R. Eyre, M. Weis, and J. Rai, unpublished observations.
      This is consistent with the low yield of the C-telopeptide aldol adduct (see Figs. 5A 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 its HHL artifactual product on LC/MS (see Fig. 8F) (
      • 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.
      ). Thus, with progressive chromatography under denaturing and acidic conditions, the labile adduct that creates HHL on acid hydrolysis continues to dissociate.
      With regard to Points 2 and 3, we explain above and in 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.
      ) why peptides prepared from collagen yield so little HHL. Such concerns do not apply to HHMD because tissue borohydride reduction quantitatively could convert the N-telopeptide dimer pool (see Fig. 5) (
      • 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.
      ) to HHMD-linked N-telopeptide to α1(I)K930 structures (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.
      ). The latter do match exactly the peptide structures shown including the mass of HHMD, as does the peptide in Fig. 7 with its cross-link (Fig. 10) (
      • 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.
      ).
      Finally, the peptides in Fig. 6 are dominant, not minor partial cleavage products, from the N-telopeptide to the helix site under the conditions we use (10% acetonitrile in the digest) to limit the range of products.

      References

        • Yamauchi M.
        • Taga Y.
        • Terajima M.
        Letter to the Editor: Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.
        J. Biol. Chem. 2019; 294: 14163
        • 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.
        J. Biol. Chem. 2019; 294 (30733334): 6578-6590
        • 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.
        Biochemistry. 1987; 26 (3651393): 3500-3509
        • 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.
        J. Biol. Chem. 1987; 262 (3624221): 11428-11434