MOLECULAR BASIS OF SELECTIVE COBALAMIN MALABSORPTION

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Humans and other mammals absorb cobalamin (Cbl, vitamin B12), an essential micronutrient, from diet via a complex interaction of Cbl-binding proteins and a specific receptor for intrinsic factor-Cbl (IFCR) in the ileal brush-border epithelium. Untreated Cbl deficiency is life-threatening in humans and most often results from disorders of Cbl absorption. The details of Cbl transcytosis by ileal enterocytes are unknown, but the pivotal importance of IFCR is exemplified by an inherited disorder of Cbl absorption known as Imerslund-Grasbeck syndrome in humans. Recently, a canine model of selective intestinal Cbl malabsorption has been described, the only known model of an IFCR defect, that appears to be very similar to Imerslund-Grasbeck syndrome. The studies proposed use this unique disease model to advance understanding of the molecular aspects of Cbl absorption and brush-border epithelial receptor expression in health and disease states. The specific aims of these investigations are to clone normal canine IFCR cDNA, to identify the IFCR mutation causing disease in affected dogs, and to define the tissue-specific and developmental regulation of normal IFCR expression. By comparison of the kinetics of IFCR expression and posttranslational modification in normal and affected dog tissues, the cellular consequences of the IFCR mutation will be defined. Using techniques of molecular biology, protein chemistry, and cell biology, studies are designed to examine the hypotheses that there are tissue-specific differences in the post-translational modification of normal IFCR and the intracellular disposition of abnormal IFCR, and that IFCR expression is differently regulated in ileal mucosa and proximal tubules. Initially, a survey of canine tissues will be made by in vitro assay of IFCR ligand-binding activity and immunoblot to determine sites of significant IFCR expression and the possible contributions of different tissues to Cbl homeostasis. Metabolic labelling, cell fractionation, and immunoprecipitation of IFCR will be used to define the cellular phenotypes of normal IFCR expression and the receptor defect in tissue explants and/or cell lines derived from normal and affected dogs. Protein and oligosaccharide studies of IFCR purified from tissues of normal and affected dog' will be done to investigate possible tissue differences in postranslational modifications of IFCR affect intracellular disposition of the abnormal IFCR of affected dogs. Standard molecular genetic approach be used to clone normal IFCR cDNA, to define the genetic mutation, and to examine IFCR mRNA in normal and affected dog tissues. This integrated approach will elucidate aspects of IFCR structure affect function and expression in ileal and renal brush-border epithelia.