Modification of Collagenous Materials and Medical Treatment, Diagnosis and Monitoring of Fibrotic Conditions

Abstract

The present invention relates to the gene PLOD2 which codes for telopeptide lysyl hydroxylase (TLH). This enzyme converts telopeptidyl Lys into telopeptidyl Hyl, that can subsequently be converted into hydroxyallysine cross-links. Collagen with hydroxyallysine cross-links shows a higher resistance to degradation by proteinases than collagen with cross-links derived from allysine. In one aspect, the invention provides methods and compositions to prepare collagenous materials with varying biodegradation rates by varying the ratio of hydroxyallysine cross-links over allysine cross-links. In another aspect, the invention provides methods and compositions to lower the ratio of hydroxyallysine cross-links over allysine cross-links in fibrotic processes, in order to obtain a collagenous network that is more easy to degrade. Furthermore, the invention provides methods to diagnose and/or monitor fibrotic processes by measuring mRNA levels of PLOD2, by measuring protein levels of the translated mRNA, and/or by measuring enzymatic activity levels of TLH. The invention also provides the description of a high through-put system facilitating the screening of antagonists of telopeptide lysyl hydroxylase.

1 - 34 . (canceled) 
     
     
         35 . A method for diagnosing and/or monitoring the occurrence or state of a fibrotic process in a mammal comprising taking a sample from said mammal, analyzing said sample to determine the expression level of a PLOD2 gene and comparing said expression level with a standard. 
     
     
         36 . The method of  claim 35  wherein the sample is analyzed by Northern blotting or a quantitative or semiquantitative polymerase chain reaction to determine the level of mRNA derived from a PLOD2 gene. 
     
     
         37 . The method of  claim 35  wherein the sample is analyzed to determine the level of PLOD2-encoded telopeptide lysyl hydroxylase using a PLOD2-specific antibody or aptamer. 
     
     
         38 . The method of  claim 35  wherein the sample is analyzed to determine the level of enzymatic activity of a PLOD2-encoded telopeptide lysyl hydroxylase using a peptide or polypeptide containing at least one hydroxylatable lysine which is a suitable substrate for PLOD2-encoded telopeptide lysyl hydroxylase. 
     
     
         39 . An assay for screening compounds or compositions to determine their effect on telopeptide lysyl hydroxylase activity comprising contacting under enzymatically functional conditions a compound or composition to be tested with a PLOD2-encoded telopeptide lysyl hydroxylase enzyme and a suitable substrate for this enzyme, and determining the level of lysyl hydroxylation of the substrate compared to the level of lysyl hydroxylation of the substrate in the absence of the compound or composition to be tested. 
     
     
         40 . The assay of  claim 39  wherein the substrate is a peptide comprising an internal lysine and not having an N-terminal serine or threonine, such as a peptide of the formula Q-L-S-Y-G-Y-D-E-K-S-T-G-G-I-S-V-P, in particular the biotin labeled substrate biotin-Q-L-S-Y-G-Y-D-E-K-S-T-G-G-I-S-V-P. 
     
     
         41 . The assay of  claim 40  further comprising separating, after said contacting of the compound or composition to be tested with a PLOD2-encoded telopeptide lysyl hydroxylase enzyme and suitable substrate for it, the substrate from the reaction mixture, reacting the substrate successively with an oxidizing agent, such as periodate, which is capable of oxidizing the hydroxyl group to an aldehyde moiety, and a hydrazide dye, and measuring the fluorescence from the substrate. 
     
     
         42 . An assay for screening compounds or compositions to determine their effect on telopeptide lysyl hydroxylase expression comprising growing cells expressing a PLOD2encoded telopeptide lysyl hydroxylase enzyme in the presence of a compound or composition to be tested and determining the level of PLOD2 expression compared to the level of PLOD2 expression in the absence of the compound or composition to be tested.