Structural and functional analysis of CRISP3 protein in relation to diseases of exocrine pancreas

Abstract

Introduction: Human cysteine rich secretory protein 3 (CRISP3), also known as specific granule protein (SGP28) is a small 28kDa protein. CRISP-3 is widely expressed in salivary glands, pancreas (particularly in pancreatic disease), lachrymal glands, prostate, myeloid precursors, human epididymis, ovary, thymus and colon. Human CRISP3 share similarity with snake venoms as well as Glioma Pathogenesis Related protein GLIPR1. Here we are reporting the extent of alignment of human CRISP3 protein with pancreatic enzymes and snake venom proteins and presence of GLIPR1 and CRISP3 genes containing conserved GHYTQVVW motifs in clinical samples of pancreatitis and pancreatic cancer. Material and Methods: Protein sequences with accession numbers for Human CRISP3, Snake Venom and enzymes present in pancreatic juices i.e. Trypsinogen, Cathepsin, Lipase, Amylase and Elastase were retrieved from swissprot, protein database. These sequences were aligned in silico for demonstrating the presence of 16 conserved cysteine residues and other motifs i.e. GENL and GHYTQVVW. The multiple sequence alignment (MSA) was performed. Presence of CRISP-3 conserved motifs in clinical samples of pancreatitis and pancreatic cancer was evaluated by PCR. DNA extracted from a small piece taken from inflamed pancreatic tail biopsy with confirmed diagnosis for pancreatitis and an excised pancreatic cancer tissue was subjected to PCR to conform presence of CRISP3 and GLIPR1 genes. Results: Our results show conservation of 9 Cysteine residues out of 16 Cysteine residues in majority of pancreatic enzymes and notable conservation of GENL and GHYTQVVW motifs across snake venom proteins. PCR yielded desired amplicon of expected size with the primers spanning conserved residues and core consensus motifs specifically designed to amplify CRISP3 gene in clinical samples of pancreatitis and pancreatic cancer. We also detected presence of GLIPR1 gene sequence in the DNA extracted from clinical samples of pancreatitis and pancreatic cancer thus confirming similarity between the pathogenesis related protein and CRISP3. Conclusion: Notable conservation of cysteine residues across pancreatic enzymes, snake venom and Human CRISP3 opens avenues of further research to evaluate the likely clinical potential of the conserved residues as putative biomarkers in exocrine pancreatic diseases. To the best of our knowledge this is the first report where primers designed around conserved GHYTQVVW motifs to amplify CRISP3 gene and its structural similarity with GLIPR1 gene sequence in clinical samples of pancreatitis and pancreatic cancer has been validated.