Mouse anti-Human HK3 monoclonal antibody, clone 5G6

The third human tissue kallikrein to be identified, hK2, could be an alternate or complementary marker to kallikrein hK3 (prostate-specific antigen) for prostate diseases, Most of the hK2 in seminal plasma forms an inactive complex with protein C inhibitor (PCI), a serpin secreted by seminal vesicles, As serpin inhibitors behave as suicide substrates that are cleaved early in the interaction with their target enzyme, and kallikreins have different sensitivities to serpin inhibitors, we prepared a series of substrates with intramolecularly quenched fluorescence based on the sequences of the serpin reactive loops, They were used to compare the substrate specificities of hK1 and hK2, which both have trypsin-like specificity, and thus differ from chymotrypsin-like hK3, The serpin-derived peptides behaved as kallikrein substrates whose sensitivities reflected the specificity of the parent inhibitory proteins, Substrates derived from PCI were the most sensitive for both hK1 and hK2 with specificity constants of about 10(7) M-1.s(-1). Those derived from antithrombin III and alpha(2)-antiplasmin were more specific for hK2 while a kallistatin-derived substrate was specifically cleaved by hK1, hK1 and hK2 substrates of greater specificity were obtained using chimeric peptides based on the sequence of serpin reactive loops, The main difference between specificities of hK1 and hK2 arise because hK2 can accommodate positively charged as well as small residues at P-2 and requires an arginyl residue at P-1, Thus, unlike hK1, hK2 does not cleave kininogen derived substrates overlapping the region of N-terminal insertion of bradykinin in human kininogens.

Background: CpG island methylator phenotype (CIMP) is found in 15-20% of malignant colorectal tumors and is characterized by strong CpG hypermethylation over the genome. The molecular mechanisms of this phenomenon are not still fully understood. The development of CIMP is followed by global gene expression alterations and metabolic changes. In particular, CIMP-low colon adenocarcinoma (COAD), predominantly corresponded to consensus molecular subtype 3 (CMS3, "Metabolic") subgroup according to COAD molecular classification, is associated with elevated expression of genes participating in metabolic pathways. Methods: We performed bioinformatics analysis of RNA-Seq data from The Cancer Genome Atlas (TCGA) project for CIMP-high and non-CIMP COAD samples with DESeq2, clusterProfiler, and topGO R packages. Obtained results were validated on a set of fourteen COAD samples with matched morphologically normal tissues using quantitative PCR (qPCR). Results: Upregulation of multiple genes involved in glycolysis and related processes (ENO2, PFKP, HK3, PKM, ENO1, HK2, PGAM1, GAPDH, ALDOA, GPI, TPI1, and HK1) was revealed in CIMP-high tumors compared to non-CIMP ones. Most remarkably, the expression of the PKLR gene, encoding for pyruvate kinase participating in gluconeogenesis, was decreased approximately 20-fold. Up to 8-fold decrease in the expression of OGDHL gene involved in tricarboxylic acid (TCA) cycle was observed in CIMP-high tumors. Using qPCR, we confirmed the increase (4-fold) in the ENO2 expression and decrease (2-fold) in the OGDHL mRNA level on a set of COAD samples. Conclusions: We demonstrated the association between CIMP-high status and the energy metabolism changes at the transcriptomic level in colorectal adenocarcinoma against the background of immune pathway activation. Differential methylation of at least nine CpG sites in OGDHL promoter region as well as decreased OGDHL mRNA level can potentially serve as an additional biomarker of the CIMP-high status in COAD.