Characterization of the prohormone complement in cattle using genomic libraries and cleavage prediction approaches.

backgroundNeuropeptides are cell to cell signalling molecules that regulate many critical biological processes including development, growth and reproduction. These peptides result from the complex processing of prohormone proteins, making their characterization both challenging and resource demanding. In fact, only 42 neuropeptide genes have been empirically confirmed in cattle. Neuropeptide research using high-throughput technologies such as microarray and mass spectrometry require accurate annotation of prohormone genes and products. However, the annotation and associated prediction efforts, when based solely on sequence homology to species with known neuropeptides, can be problematic.

resultsComplementary bioinformatic resources were integrated in the first survey of the cattle neuropeptide complement. Functional neuropeptide characterization was based on gene expression profiles from microarray experiments. Once a gene is identified, knowledge of the enzymatic processing allows determination of the final products. Prohormone cleavage sites were predicted using several complementary cleavage prediction models and validated against known cleavage sites in cattle and other species. Our bioinformatics approach identified 92 cattle prohormone genes, with 84 of these supported by expressed sequence tags. Notable findings included an absence of evidence for a cattle relaxin 1 gene and evidence for a cattle galanin-like peptide pseudogene. The prohormone processing predictions are likely accurate as the mammalian proprotein convertase enzymes, except for proprotein convertase subtilisin/kexin type 9, were also identified. Microarray analysis revealed the differential expression of 21 prohormone genes in the liver associated with nutritional status and 8 prohormone genes in the placentome of embryos generated using different reproductive techniques. The neuropeptide cleavage prediction models had an exceptional performance, correctly predicting cleavage in more than 86% of the prohormone sequence positions.

conclusionA substantial increase in the number of cattle prohormone genes identified and insights into the expression profiles of neuropeptide genes were obtained from the integration of bioinformatics tools and database resources and gene expression information. Approximately 20 prohormones with no empirical evidence were detected and the prohormone cleavage sites were predicted with high accuracy. Most prohormones were supported by expressed sequence tag data and many were differentially expressed across nutritional and reproductive conditions. The complete set of cattle prohormone sequences identified and the cleavage prediction approaches are available at http://neuroproteomics.scs.uiuc.edu/neuropred.html.