Delivery of Vitamin B12 to Human Lymphocytes by Transcobalamins I, II and III

Abstract

It has been established that three binders of vitamin B,, can be separated by DEAEcellulose column chromatography following addition of labelled vitamin to normal serum (1-4). All 3 binders are present in trace amounts. They are known as transcobalamin I (TC I ) , transcobalamin I1 (TC 11), and transcobalamin I11 (TC 111) or "main protein peak binder" (MPPB) . TC I1 and TC I11 have an electrophoretic mobility of a-globulin, while TC I has the mobility of ,&globulin (1, 2).Labelled vitamin B,, is cleared rapidly from intravenously administered TC I1 5 7 C~ B,, but much more slowly from TC I 5 7 C~ B,, (2,5,6). The differences in clearance are less marked, however, when the transcobalamins are labelled with orally administered labelled vitamin BI2. The rapid clearance of 57C0 B12 from intravenously administered B,,--TC I1 has been attributed to partial denaturation of the protein during its purification in vitro (7). Finkler and Hall (8) reported that HeLa cells take up TO B,, from TC I1 in vitro but not from TC I while Retief et al. (9) observed that TC I1 delivers vitamin B,z to reticulocytes at a faster rate than TC I. Uptake by perfused rat liver of labelled vitamin B,, from 5 7 C~ B,, labelled TC I1 and TC I11 from serum of normal donors and pernicious anemia patients was substantially greater than from 5 7 C~ B,, labelled TC I (10). The exact role of TC 111 vita-1In receipt of a grant from the Leukemia Research Fund.747 min B,, in plasma is still uncertain. Chanarin and co-workers have recently shown that following oral administration of 5 7 C~ BIZ, the labelled vitamin is taken up simultaneously by all 3 serum binders (7, 11). These authors also showed immunological identity of TC I and TC 111. However, the ability of TC I11 to deliver vitamin B,, to cells is still unknown.H o a r a n d et al. (12) used phytohemagglutinin (PHA) -transformed lymphocytes as a model cell system to investigate uptake of serum bound radioactive vitamin B,, by normal proliferating human hemopoietic cells. In the present study we have used this system to compare the ability of human TC I11 with that of TC I and TC I1 to deliver labelled vitamin B,, to human cells. Materials and Methods. Preparation of vitamin B,, binders.This has been reported in detail elsewhere (13). Thirty ml of normal serum were used in each column chromatographic separation, Serum used in each experiment was drawn from an individual donor considered to be hematologically normal. Five hundred pg of 5 7 C~ B,, (specific activity 120 mCi/mg; purchased from the Radiochemical Centre, Amersham, Amersham, England) were added per ml to 30 ml of serum. The solution was allowed to stand at 37" for 20 min, then dialysed at 4" against 4 litres of 0.0175 M sodium phosphate buffer, pH 6.3, for 48 hr. The buffer was changed twice during this period.DEAE-cellulose ( Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 and 0.95 mEq/g) was packed after

Cellular uptake of cobalamin: Transcobalamin and the TCblR/CD320 receptor

“…The receptors appear to be segregated in discrete microvilli and appear to be directed to clathrin coated pits during endocytosis (92, 93). The expression of TCblR appears to be cell cycle associated with most receptor expression during log phase of growth in actively proliferating cells and drastically down-regulated in resting cells (94-96). The existence of this membrane attached receptor was known for more that half a century but the purification and structural characterization of this protein proved to be difficult due to the low amounts of the receptor and the ligand specific affinity chromatography required to purify the protein.…”

A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations.

“…By contrast, congenital TCII deficiency in humans leads to severe manifestations of B12 deficiency; thus, TCII appears necessary for transport of B12 from the gut to peripheral tissues (9,10). A number of studies have demonstrated that TCII promotes the uptake of B12 by human cells in vitro (11)(12)(13)(14). Recently, specific saturable binding sites for rat TCII-B12 complexes on rat liver plasma membranes have been described (15).…”

Role of Transcobalamins I, II and III in the Transfer of Vitamin B₁₂ to Human Bone Marrow Cells <i>in vitro</i>

“…However, as the present data on human bone marrow cells indicate that the mechanism of uptake of vitamin B12 from TCI and TCIII is different from that of the uptake of unbound vitamin B12, the results of M eyer et al [16] may have to be re-interpreted as indicating that all 3 transcobalamins are capable of transferring vitamin B12 to stimulated lymphocytes in vitro.…”

“…In a recent study, M eyer et al [16] measured the uptake of vitamin B12 by PHA-stimulated human lymphocytes, found that the uptake me diated by TCI or TCIII was quantitatively similar to the uptake of un bound vitamin B12 and concluded that TCI and TCIII played no role in the transfer of vitamin BI2 to tissue cells. However, as the present data on human bone marrow cells indicate that the mechanism of uptake of vitamin B12 from TCI and TCIII is different from that of the uptake of unbound vitamin B12, the results of M eyer et al [16] may have to be re-interpreted as indicating that all 3 transcobalamins are capable of transferring vitamin B12 to stimulated lymphocytes in vitro.…”