title: Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly.
abstract: 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 alpha-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 cross-link site of the alpha1alpha1alpha2 network was identified, on the basis of x-ray crystal structures at 1.9-A resolution, in which the side chains of Met93 and Lys211 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., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612); however, at the higher resolution of 1.5 A, we found no evidence for this cross-link (Vanacore, R. M., Shanmugasundararaj, S., Friedman, D. B., Bondar, O., Hudson, B. G., and Sundaramoorthy, M. (2004) J. Biol. Chem. 279, 44723-44730). Given this discrepancy in crystallographic findings, we sought chemical evidence for the location and nature of the reinforcement/cross-link site. Trypsin digestion of monomer and dimer subunits excised a approximately 5,000-Da complex that distinguished dimers from monomers; the complex was characterized by mass spectrometry, Edman degradation, and amino acid composition analyses. The tryptic complex, composed of two peptides of 44 residues derived from two alpha1 NC1 monomers, contained Met93 and Lys211 post-translationally modified to hydroxylysine (Hyl211). Truncation of the tryptic complex with post-proline endopeptidase reduced its size to 14 residues to facilitate characterization by tandem mass spectrometry, which revealed a covalent linkage between Met93 and Hyl211. The novel cross-link, termed S-hydroxylysyl-methionine, reflects at least two post-translational events in its formation: the hydroxylation of Lys211 to Hyl211 within the NC1 domain during the biosynthesis of alpha-chains and the connection of Hyl211 to Met93 between the trimeric NC1 domains of two adjoining triple-helical protomers, reinforcing the stability of collagen IV networks.
authors.affiliation: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160-7421, USA.
authors.forename: Roberto M
authors.initials: RM
authors.lastname: Vanacore
authors.forename: David B
authors.initials: DB
authors.lastname: Friedman
authors.forename: Amy-Joan L
authors.initials: AJ
authors.lastname: Ham
authors.forename: Munirathinam
authors.initials: M
authors.lastname: Sundaramoorthy
authors.forename: Billy G
authors.initials: BG
authors.lastname: Hudson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D024141:Collagen Type IV / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D006901:Hydroxylysine / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D024141:Collagen Type IV; D003432:Cross-Linking Reagents; D006901:Hydroxylysine; D011392:Proline; D008715:Methionine; D014357:Trypsin; D008239:Lysine; D004229:Dithiothreitol