Production of gastric intrinsic factor, transcobalamin, and haptocorrin in opossum kidney cells

Abstract

Opossum kidney epithelial cells were shown previously to synthesize and secrete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis that renal tubular cells also produce intrinsic factor (IF), and this production provides an explanation for the presence of IF in urine. By using antisera raised against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein was blocked by recombinant human IF. When proteins secreted into the media were separated electrophoretically under nondenaturing conditions after binding with [(57)Co]Cbl, a broad major band migrated at a relative front independently of recombinant IF or TCII, and probably represents Hc, as the Cbl binding is blocked by cobinamide. Small amounts of bound [(57)Co]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The presence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming the ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragment of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western blot confirmed the presence of IF protein. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other epithelia.

Loss of genes implicated in gastric function during platypus evolution

“…The gene encoding gastric intrinsic factor ( GIF ), which is necessary for the absorption of vitamin B 12 , is perfectly conserved in platypus. This protein is secreted by chief or parietal cells in most eutherians, but it is mainly produced by pancreatic cells in dogs as well as in opossum, in which no gastric expression can be detected [ 28 , 29 ]. It is therefore likely that the expression of this gene was pancreatic before the prototherian-therian split, and the intrinsic factor might still be secreted by the pancreas in platypus, where it can exert its physiological function.…”

The kidney in vitamin B12and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins

“…The mechanism, by which reabsorbed B 12 is released from the tubular cells following reabsorption, is largely unknown. Both TC and HC, and possibly IF, are synthesized in proximal tubule cells in vitro (19,104,112). TC mRNA was identified in adult porcine and human kidney, whereas HC mRNA could not be identified in the adult porcine kidney (104).…”