1. Volume 24, Issue 10, Pages 1276-1284.e3 (October 2017)
Hydroxyhomocitrulline Is a Collagen-Specific Carbamylation Mark that Affects Cross-link Formation 
Yuki Taga, Keisuke Tanaka, Chieko Hamada, Masashi Kusubata, Kiyoko Ogawa-Goto, Shunji Hattori 
Cell Chemical Biology 
Volume 24, Issue 10, Pages e3 (October 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 1276-1284. e3DOI: (10. 1016/j. chembiol
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3. Figure 1 Mass Spectrometric Identification of HHCit in Collagen
MS/MS spectra of a (A) Hyl standard, (B) HHCit standard, and (C) HHCit purified from skin collagen from an 8-year-old bovine. *Unknown peaks probably originating from contaminated compounds. MRM chromatograms of HCit and HHCit in acid hydrolysates of (D) the bovine collagen, (E) bovine elastin, and (F) bovine serum albumin.
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4. Figure 2
Age-Related Alterations in Carbamylation Rates of HCit and HHCit in Tissue Collagens
Collagens purified from (A) skin, (B) bone, and (C) tail tendon of Sprague-Dawley rats (0.5, 1, 2, 3, 6, 12, and 18 months of age) were subjected to acid hydrolysis with carbamylated SI-collagen used as an internal standard. Generated amino acids were quantitated by LC-MS in MRM mode. Carbamylation rates of HCit and HHCit are expressed as mmol/mol Lys and mmol/mol Hyl, respectively. The data represent the mean ± SD (N = 3).
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5. Figure 3 In Vivo and In Vitro Acceleration of Carbamylation
(A) Experimental design for in vivo carbamylation using mice. Five groups (I–V) of ICR mice received water or 15 mM sodium cyanate in water (SC-water) with the schedule shown in the schematic diagram.
(B) HCit and HHCit levels were quantitated by LC-MS for collagens purified from skin, bone, and tail tendon of mice in each group. The data represent the mean ± SD (N = 4). (C–D) Collagens purified from skin, bone, and tail tendon of normal mice were subjected to in vitro carbamylation. HCit and HHCit levels were quantitated by LC-MS for collagens carbamylated with (C) 0.1 M or (D) 0.01 M potassium cyanate at 37°C for 2, 6, and 24 hr. The data represent the mean ± SD (N = 3).
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6. Figure 4
Pathways of Collagen Cross-Linking and Possible Inhibition of the Cross-Link Formation by Carbamylation
(A) Under physiological conditions, collagen cross-linking occurs in tissues in following consecutive steps. Firstly, the ɛ-amino group of Lys or Hyl at nontriple helical domains (N- and C-telopeptides) is converted into an aldehyde by LOX. Secondly, the generated aldehyde couples with the ɛ-amino group of Lys or Hyl at the triple helical domain by Schiff base formation to form immature divalent cross-links. Lastly, the intermediate cross-links mature into trivalent or tetravalent cross-links with additional involvement of Lys, Hyl, or His (Yamauchi and Sricholpech, 2012).
(B) Carbamylation of Lys or Hyl at helical cross-linking sites blocks the cross-link formation, because the carbamylated residue cannot react with the telopeptidyl aldehyde.
(C) Carbamylation of telopeptidyl Lys or Hyl also blocks the cross-link formation, because LOX cannot oxidize the carbamylated residue to generate an aldehyde.
Cell Chemical Biology  , e3DOI: ( /j.chembiol )
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