Characterization of collagenous peptides bound to lysyl hydroxylase isoforms.
Lysyl hydroxylase (LH, EC 1.14.11.4) is the enzyme catalyzing the formation of hydroxylysyl residues in collagens and other proteins with collagenous domains. Although lower species, such as Caenorhabditis elegans, have only one LH orthologue, LH activity in higher species, such as human, rat, and mouse, is present in three molecules, LH1, LH2, and LH3, encoded by three different genes. In addition, LH2 is present in two alternatively spliced forms (LH2a, LH2b). To understand the functions of the four molecular forms of LH in vertebrates, we analyzed differences in the binding and hydroxylation of various collagenous peptides by the LH isoforms. Nine-amino acid-long synthetic peptides on Pepspot were used for the binding analysis and an activity assay to measure hydroxylation. Our data with 727 collagenous peptides indicated that a positive charge on the peptide and specific amino acid residues in close proximity to the lysyl residues in the collagenous sequences are the key factors promoting peptide binding to the LH isoforms. The data suggest that the LH binding site is not a deep hydrophobic pocket but is open and hydrophilic where acidic amino acids play an important role in the binding. The data do not indicate strict sequence specificity for the LH isoforms, but the data indicated that there was a clear preference for some sequences to be bound and hydroxylated by a certain isoform.
Risteli
Maija
M
Department of Biochemistry, Biocenter Oulu, University of Oulu, P. O. Box 3000, Fin-90014, University of Oulu, Finland.
Niemitalo
Olli
O
Lankinen
Hilkka
H
Juffer
André H
AH
Myllylä
Raili
R
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D007527:Isoenzymes; D010455:Peptides; D003094:Collagen; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase