Specificity of trypsin and carboxypeptidase B for hydroxylysine residues in denatured collagens

Wu, George Y.; Pereyra, Bolivar; Seifter, Sam

doi:10.1021/bi00518a013

Cited by 15 publications

(15 citation statements)

References 19 publications

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“…These results extend the findings of Wu et al (6) who found that glycosylated Hyl in collagen was an unacceptable substrate for trypsin, but that the -OH group, by itself, was insufficient to prevent proper approximation of enzyme for substrate. Our decameric peptide model showed similar results with the caveat that Hyl proved to be less susceptible than Lys to peptide bond hydrolysis by the two serine proteases we investigated.…”

Section: Methodssupporting

confidence: 89%

Hydroxylation of Lys Residues Reduces Their Susceptibility to Digestion by Trypsin and Lysyl Endopeptidase

Molony¹,

Quan²,

Mulkerrin³

et al. 1998

Analytical Biochemistry

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Section: Methodssupporting

confidence: 89%

Hydroxylation of Lys Residues Reduces Their Susceptibility to Digestion by Trypsin and Lysyl Endopeptidase

Molony¹,

Quan²,

Mulkerrin³

et al. 1998

Analytical Biochemistry

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“…In the dimer, the Hyl 211 -Ala 212 bond is resistant to trypsin cleavage, resulting in the excision of the T-1414 peptide with an intact Hyl 211 -Ala 212 bond (Fig. 5); such bonds are known to be susceptible to trypsin cleavage except when Hyl is modified by a carbohydrate unit (14). This poses the questions of whether the Hyl 211 -Ala 212 bond is resistant or susceptible to cleavage when present in the monomer, and , a structure that is incompatible with all the mass spectrometry results reported herein.…”

Section: Evidence For a Covalent Cross-link In The Tryptic Complex (Tmentioning

confidence: 99%

Identification of S-Hydroxylysyl-methionine as the Covalent Cross-link of the Noncollagenous (NC1) Hexamer of the α1α1α2 Collagen IV Network

Vanacore

Friedman

Ham

et al. 2005

Journal of Biological Chemistry

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Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three ␣-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the crosslink site of the ␣1␣1␣2 network was identified, on the basis of x-ray crystal structures at 1.9-Å resolution, in which the side chains of Met 93 and Lys 211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P. Collagen IV networks are components of basement membranes that underlie epithelia, compartmentalize tissue, and influence cell behavior. The networks are assembled from a family of six polypeptide chains (␣1-␣6) that associate forming three subtypes of triple-helical protomers with distinct chain compositions: ␣1␣1␣2, ␣3␣4␣5, and ␣5␣5␣6 (1, 2). The protomers self-assemble by end-to-end associations in which the amino termini of four protomers associate tail-to-tail forming the 7 S domain, and the carboxyl termini of two protomers associate head-to-head through the noncollagenous (NC1) 1 domains, forming dimers. At the interface of the head-to-head connection, the trimeric NC1 domains exist as a hexamer, a stable complex that can be excised by cleavage with collagenase for in vitro studies.The NC1 domain plays a pivotal role in the assembly of the distinct collagen IV networks. In protomer assembly, the NC1 domains (monomers) of three chains interact, forming a NC1 trimer, to select and register chains for triple-helix formation. In the network assembly, the NC1 trimers of two protomers interact, forming a NC1 hexamer structure, to select and connect protomers. Upon exposure to acidic pH or denaturants, isolated NC1 hexamer dissociates into monomers and dimers, the latter reflecting the presence of cross-links that stabilize the trimer-trimer interface. The cross-links connect ␣1-like monomers (␣1-␣1, ␣1-␣5, and ␣3-␣5) and ␣2-like monomers (␣2-␣2, ␣2-␣6, and ␣4 -␣4) (3, 4). For two decades, the reduc- 1 The abbreviations used are: NC1, non-collagenous domain; PBM, placenta basement membrane; GdnHCl, guanidine hydrochloride; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; LC-ESI/MS/MS and LC-ESI/MS 3 , liquid chromatography electrospray tandem mass spectrometry; HPLC, high performance liquid chromatography; DTT, dithiothreitol; Hyl, hydroxylysine; MS 3 , third fragmentation mass spectrometry.

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“…More recently, a system has been found in liver for phosphorylation of free hydroxylysine by a hydroxylysine kinase that utilizes GTP as the phosphono donor (4,5). That reaction is the first step in the complete degradative metabolism of hydroxylysine liberated by digestion of collagenous proteins as, for instance, shown by our laboratory, by the combined actions of trypsin and carboxypeptidase B (6).…”

mentioning

confidence: 91%

Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

Urushizaki¹,

Seifter²

1985

Proc. Natl. Acad. Sci. U.S.A.

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05-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCO without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32p was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3Hllysine into hydroxylysine residues of cell-associated collagen and then 32p into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32p was transferred from [y-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.With the discovery of proteins containing phosphotyrosine residues (1, 2), all of the coded hydroxy amino acids inserted into polypeptide chains have been shown to undergo posttranslational phosphorylation by action of protein kinases and ATP. Two other hydroxy amino acids are in themselves formed by post-translational hydroxylation reactions; these are hydroxyproline in collagens and elastins, formed from proline residues, and 5-hydroxylysine in collagens, formed from lysine residues. We have begun studies of the phosphorylation of hydroxylysine and hydroxyproline residues in proteins.In its free form, 05-phosphohydroxylysine was identified tentatively in the extracellular fluids of bovine embryos in the early screening applications of paper chromatography (3). More recently, a system has been found in liver for phosphorylation of free hydroxylysine by a hydroxylysine kinase that utilizes GTP as the phosphono donor (4, 5). That reaction is the first step in the complete degradative metabolism of hydroxylysine liberated by digestion of collagenous proteins as, for instance, shown by our laboratory, by the combined actions of trypsin and carboxypeptidase B (6).For many years, we have been concerned with possible functions for hydroxylysine residues. It is known that such residues, like those of lysine, can undergo oxidation at C-6 to form corresponding aldehyde groups, which can participate in crosslink formation (7). The hydroxyl groups of hydroxylysine residues can undergo glycosylation by action of a transferase and UDP-galactose, and the substituted galactose residue c...

show abstract

Specificity of trypsin and carboxypeptidase B for hydroxylysine residues in denatured collagens

Cited by 15 publications

References 19 publications

Hydroxylation of Lys Residues Reduces Their Susceptibility to Digestion by Trypsin and Lysyl Endopeptidase

Hydroxylation of Lys Residues Reduces Their Susceptibility to Digestion by Trypsin and Lysyl Endopeptidase

Identification of S-Hydroxylysyl-methionine as the Covalent Cross-link of the Noncollagenous (NC1) Hexamer of the α1α1α2 Collagen IV Network

Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

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