The chemistry of the collagen cross-links. Characterization of the products of reduction of skin, tendon and bone with sodium cyanoborohydride

Reduction of tissues with sodium cyanoborohydride at pH7.4 gave results identical with those obtained by KBH4 treatment. On reduction with sodium cyanoborohydride at pH 4.4, however, a previously undetected basic compound was formed and was identified by mass spectrometry and chemical degradation techniques as dihydrohydroxymerodesmosine. Histidino-hydroxymerodesmosine was not present, and further analysis confirmed that reduced aldol, a mojor product of reduction with KBH4 at the lower pH, was also absent. These results, together with an analysis of the time course of the reduction, support previous assertions that histidino-hydroxymerodesmosine is an artifact [robins *Bailey (1973) Biochem. J. 135, 657-665] and suggests that the non-reduced form of hydroxymerodesmosine probably does not constitute a major intermolecular bond in vivo.

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The chemistry of the collagen cross-links. The characterization of Fraction C, a possible artifact produced during the reduction of collagen fibres with borohydride

Biochemical Journal ◽

10.1042/bj1350657 ◽

1973 ◽

Vol 135 (4) ◽

pp. 657-665 ◽

Cited By ~ 44

Author(s):

Simon P. Robins ◽

Allen J. Bailey

Keyword(s):

Aldol Condensation ◽

Condensation Product ◽

Reduced Form ◽

Borohydride Reduction ◽

Cross Link ◽

Collagen Fibres ◽

Isolation And Identification ◽

Cross Links

The present paper describes the isolation and identification of a major radioactive component of borotritide-reduced collagen, previously designated Fraction C. The derived structure for the compound confirms that it is identical with the ‘post-histidine’ component described by Tanzer et al. (1973) and given the trivial name histidino-hydroxymerodesmosine. Detailed studies of the effects of acid pH on the formation of Fraction C after borohydride reduction demonstrated the apparent lability of the non-reduced form, thus confirming our previous findings (Bailey & Lister, 1968). Inhibition of the formation of this component by the acid treatment appears to be due to protonation of the histidine imidazole group. Since the only new component formed on reduction of the acid-treated fibres was the reduced aldol condensation product, these results indicate that neither the histidine nor the hydroxylysine residues can be involved in covalent linkage with the aldol condensation product in the native fibre. It is suggested therefore that the proposed non-reduced aldimine form of Fraction C does not exist as an intermolecular cross-link in vivo. Thus the presence of histidino-hydroxymerodesmosine as a tetrafunctional cross-link in reduced collagen fibres is a result of a base-catalysed reaction promoted by the borohydride-reduction procedure and this component must therefore be considered as an artifact.

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Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

Biochemical Journal ◽

10.1042/bj1170819 ◽

1970 ◽

Vol 117 (5) ◽

pp. 819-831 ◽

Cited By ~ 146

Author(s):

A. J. Bailey ◽

Catherine M. Peach ◽

L. J. Fowler

Keyword(s):

Mass Spectrometry ◽

Large Scale ◽

High Resolution Mass Spectrometry ◽

Collagen Fibre ◽

Reduced Form ◽

Cross Link ◽

Isolation And Characterization ◽

Cross Links

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N∈-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, ∈-hydroxynorleucine, together with its acid degradation product ∈-chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.

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Signal Transduction in Cerebral Arteries after Subarachnoid Hemorrhage—A Phosphoproteomic Approach

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.78 ◽

2013 ◽

Vol 33 (8) ◽

pp. 1259-1269 ◽

Cited By ~ 14

Author(s):

Benjamin L Parker ◽

Martin Røssel Larsen ◽

Lars IH Edvinsson ◽

Gro Klitgaard Povlsen

Keyword(s):

Mass Spectrometry ◽

Subarachnoid Hemorrhage ◽

Time Course ◽

Cerebral Arteries ◽

Delayed Cerebral Ischemia ◽

Calcium Calmodulin Dependent ◽

Intracellular Signals ◽

Extracellular Regulated Kinase ◽

Signaling Components

After subarachnoid hemorrhage (SAH), pathologic changes in cerebral arteries contribute to delayed cerebral ischemia and poor outcome. We hypothesize such changes are triggered by early intracellular signals, targeting of which may prevent SAH-induced vasculopathy. We performed an unbiased quantitative analysis of early SAH-induced phosphorylations in cerebral arteries and evaluated identified signaling components as targets for prevention of delayed vasculopathy and ischemia. Labeled phosphopeptides from rat cerebral arteries were quantified by high-resolution tandem mass spectrometry. Selected SAH-induced phosphorylations were validated by immunoblotting and monitored over a 24-hour time course post SAH. Moreover, inhibition of key phosphoproteins was performed. Major SAH-induced phosphorylations were observed on focal adhesion complexes, extracellular regulated kinase 1/2 (ERK1/2), calcium calmodulin-dependent kinase II, signal transducer and activator of transcription (STAT3) and c-Jun, the latter two downstream of ERK1/2. Inhibition of ERK1/2 6-hour post SAH prevented increases in cerebrovascular constrictor receptors, matrix metalloprotease-9, wall thickness, and improved neurologic outcome. STAT3 inhibition partially mimicked these effects. The study shows that quantitative mass spectrometry is a strong approach to study in vivo vascular signaling. Moreover, it shows that targeting of ERK1/2 prevents delayed pathologic changes in cerebral arteries and improves outcome, and identifies SAH-induced signaling components downstream and upstream of ERK1/2.

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