Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line

Abstract

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

Receptor‐mediated endocytosis of the intrinsic factor—cobalamin complex in HT 29, a human colon carcinoma cell line

“…2). We have also observed such an increase, after the same culture time, for the synthesis of transcobalamin II by HT 29 cells [4]. This delay corresponded to the time needed by the cells to reach eonfluency [6].…”

“…The receptor is located only in the distal ileum of adult humans, but it is also detected in the jejunum and in the colon of fetus, before 25 weeks of gestational age [2,3]. Recently, we have reported the synthesis of transcobalamin II and of a receptor for IF-Cbl in HT 29 cells [4,5]. HT 29 is a cell line, derived from a human colon carcinoma, which shares the same brush border enzymes than fetal cells and grows as polarized monolayers in culture [6].…”

Vitamin B12 Transporters

“…In contrast, Ramanujam and coworkers observed that Caco-2 cells only secrete TcII into the basal medium of transwell cultures with no apparent secretion of VB 1 2 -binders into the apical medium (Ramanujam et al, 1991b). In contrast, Hc was found to be secreted into the cell medium of HT-29 cell cultures (Schohn et al, 1991).…”

“…Secretion (at least from fibroblasts) is blocked by cycloheximide and puromycin (Green et al, 1976). A TCII-like molecule is synthesized and secreted from HT 29 cells and Caco-2 cells (Schohn et al, 1991;Dix et al, 1990). The protein has a molecular weight of 44 kDa with an isoelectric point of 6.4, and is immunologically cross-reactive with human TCII (Tables I and II) (Schohn et al, 1991).…”

“…IFR expression and functional activity has been found on the polarized cells of intestinal origin such as the human colon carcinoma cell lines Caco-2 Ramanujam et al, 1991a;Hassan and Mackay, 1992; and HT29 (Guéant et al, 1992;Schohn et al, 1991), Expression has also been observed in two kidney lines, Opossum Kidney and the LLCPK1 line from porcine kidney (Ramanujam et al, 1991a(Ramanujam et al, , 1994. When these cells are cultured in vitro on permeable membranes they form a confluent monolayer with tight junctions between neighboring cells, which restricts nonspecific transport of small macromolecules.…”