Abstract
Rabbits injected with pure human placental transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, transcobalamin II, in their plasma. Exogenously added transcobalamin II-[57Co]cyanocobalamin bound very poorly (2-5%) to the affected rabbit liver, kidney, and intestinal total or intestinal basolateral membrane extracts relative to the binding by membrane extracts from normal rabbit tissues. The activity was restored to normal values following a wash of affected rabbit tissue membranes with pH 3 buffer containing 200 mM potassium thiocyanate. Immunoblot analysis of normal and affected rabbit kidney and liver total membranes revealed similar amounts of 124-kDa TC II-R dimer protein. The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II-[57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl-dependent enzymes. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes. Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and thus cause severe Cbl deficiency and the noted failure to thrive.
Highlights
Rabbits injected with pure human placental transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, transcobalamin II, in their plasma
The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II-[57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl-dependent enzymes
We suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and cause severe Cbl deficiency and the noted failure to thrive
Summary
Vol 271, No 8, Issue of February 23, pp. 4195–4200, 1996 Printed in U.S.A. In Vitro and in Vivo Inactivation of Transcobalamin II Receptor by Its Antiserum*. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and cause severe Cbl deficiency and the noted failure to thrive. The consequence of defective uptake of plasma TC II-Cbl due to functional loss of TC II-R should result in the faster development of Cbl deficiency, since TC II-R-mediated uptake of TC II-Cbl is the only mode of delivery of physiological amounts of Cbl to all the tissues [10] This hypothesis was validated in rabbits that were injected with human TC II-R for the purposes of raising polyclonal antibodies to TC II-R. The results of the current study show that human TC II-R antibodies inhibit both in vivo and in vitro the binding of TC II-Cbl in effect creating functional loss of TC II-R activity, suppressing Cbl transport, development of intracellular Cbl deficiency, and the noted failure to thrive
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