James A. Begley

Synthesis and secretion of transcobalamin II by cultured astrocytes derived from human brain tissue.

Astrocytes derived from human brain tissue secreted a single cobalamin (vitamin B12, Cbl) binding protein over a 4 day period in culture. Cycloheximide reversibly inhibited the release, and the binding protein was identified as transcobalamin II (TCII) based on molecular size, reaction with anti-human TCII antiserum, precipitation with 2.0 M ammonium sulfate and its ability to bind radioactive cyanocobalamin. It also enhanced the cellular incorporation of the vitamin. Our data show that cultured cells from human brain synthesize and secrete TCII and suggests that at least some of the TCII known to be present in cerebrospinal fluid may originate from within the central nervous system.

The methylcobalamin metabolism of cultured human fibroblasts

The effect of supplying exogenous methylcobalamin (MeCbl), a methyl donor to methionine synthase (MS), on the cellular metabolism of MeCbl was tested in cultured fibroblasts from healthy persons and from a subject with an inherited defect in the synthesis of MeCbl. MeCbl bound to transcobalamin II (TCII) was taken up in larger amounts than cyanocobalamin (CN-Cbl), but was equal to the uptake of hydroxocobalamin (OH-Cbl). The form of Cbl in the lysosomes persisted as the same form, bound to TCII, to which the cells were exposed in the medium. Once released from the lysosomes, both MeCbl and OH-Cbl were converted in the same proportions to coenzyme forms, suggesting equivalent entry into common cellular pools of Cbl from which active forms are synthesized. Exogenous MeCbl enjoyed no advantage in binding to MS, in synthesis of MeCbl, and in supporting cell division in the absence of methionine. All evidence supported the concept that in human cells the active MeCbl on MS forms de novo on the enzyme. It appeared unlikely that therapeutic MeCbl would have any advantage over OH-Cbl in the treatment of MeCbl deficiency or Cbl deficiency in general.

Methionine dependency of cultured human lymphocytes.

Abstract Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.

The role of cobalamin in synthesis of protein and immunoglobulins by human peripheral lymphocytes

Abstract The effects of vitamin B 12 (cobalamin, Cb1) on IgG synthesis were measured in human peripheral blood lymphocytes (PBL) cultured for 6 days in the presence of pokeweed mitogen. The synthesis and release of total protein and IgG were measured by precipitation of the proteins in the media that had incorporated 3 H leucine. Cells from 5 Cbl deficient donors synthesized and released less protein and IgG than cells from healthy donors. Large amounts of free Cbl added in vitro enhanced the synthetic capacities of the Cbl deficient cells but not the normal cells. Cells from 6 healthy donors were somewhat depleted of Cbl in vitro by culture in the absence of any source of Cbl. When Cbl was added there was a significant enhancement of protein synthesis, but the increase in IgG synthesis, while considerable, did not reach significance. No effect of deficiency of Cbl or the addition of Cbl was observed on cell replication or blastogenesis. Apparently Cbl has an effect on protein and IgG synthesis and release by human PBL in addition to any potential effect on DNA synthesis and cell division.

A radioimmunoassay for the R-type binders of cobalamin

A radioimmunoassay of R-type binders of cobalmin was devised and tested. The assay incorporated an antibody against purified human salivary R binder as the binding reagent. The labeled ligand was cyano[57Co]cobalamin bound to the R binder of pooled human saliva. The standard source of unlabeled ligand was also from human saliva of known R binder content. The assay was responsive to R binders of several sources, to either pure R or that of crude sources and equally to R binder saturated or unsaturated with cobalamin. It was not responsive to transcobalamin II. The assay was reproducible and reliable.

Atypical cobalamin binding in the serum of congenital deficiency of transcobalamin II

The serum cobalamin (Cb1) binding patterns were described in nine children with congenital deficiency of transcobalamin II (TC II). Immunoreactive TC II was < 100 pg/ml TC II‐Cb1 equivalent in eight and 150 pg/ml in the ninth. There was neither endogenous TC II‐Cb1 (holo TC II) nor apo TC II. Thus, the defect was characterized by the absence of any binding of Cb1 to TC II either in vivo or in vitro and either non‐detectable or much reduced immunoreactive TC II. Only the serum from an untreated infant bound any added Cb1 at all, but in every sera there was binding of endogenous Cb1 to a substance of the molecular size of albumin. Native Cb1 was also bound to R binder and in some instances was incorporated into large complexes. The precise nature, cause and consequences of this atypical binding are unknown.

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Abstract Human serum transcobalamin II (TC II), a vitamin B 12 (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[ 57 Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[ 57 Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.

The role of transcobalamin II in the methionine dependency of human lymphocytes.

Abstract Neither normal human B lymphoblasts (RPMI 6410) transformed by the EB virus nor human peripheral blood lymphocytes (PBL) stimulated by a mitogen replicated well when the methionine (Met) of the medium was replaced with homocysteine (Hcy). Cbl bound to human transcobalamin II (TC II) substantially increased cell division over that observed when the Cbl of the medium was in the free form. Although, as expected, the TC II enhanced the cell entry of Cbl 1000-fold, this was not the basis of the TC II effect. Through adjustment of the respective concentrations of free Cbl and TC II-Cbl in the medium, equal amounts of Cbl entered the cell, yet the TC II effect persisted. TC II-Cbl did not restore cell division in the absence of Met by virus-transformed lymphoblasts from a child with defective Met synthesis from Hcy. The TC II did not act by enhanced induction of the Cbl-dependent methionine synthase activity of cell extracts but the ability of intact cells to produce Met from Hcy by the Cbl-dependent process appeared to have a role in the TC II effect.

Transcobalamin II mediated delivery of albumin-bound hydroxocobalamin to human liver cells.

Abstract We show that hydroxocobalamin bound to human serum albumin can dissociate and bind to transcobalamin II present in serum. Human liver cells in culture exposed to hydroxocobalamin bound to albumin incorporated less of the vitamin than when similar amounts of unbound hydroxocobalamin or cyanocobalamin were present. In the presence of transcobalamin II, a 4.5-fold increase in cellular uptake occurred, but this amount was less than when hydroxocobalamin or cyanocobalamin were added to transcobalamin II. These results indicate that albumin, by binding hydroxocobalamin, can alter the dynamics of binding to transcobalamin II and the subsequent cellular incorporation of this form of the vitamin.

Hydrophobic Interactions of Transcobalamin II (TC II) from Mammalian Sera

Abstract The hydrophobic properties of mammalian transcobalamin lis (TC II) were studied by chromatography of radioactive cyanocobalamin (CN[57Co]Cbl)-labeled serum on phenyl-Sepharose CL-4B. Mammalian holo TC IIs (CN[57Co]Cbl-TC II) exhibited species variability in their affinity for the hydrophobic matrix in the order dog > mouse > human > rat > rabbit. Phenyl-Sepharose chromatography of the isolated CN[57Co]Cbl-TC II peaks from gel filtration of dog and rat serum showed no hydrophobic change in dog TC II, but an increase in hydro-phobicity of rat TC II. Phenyl-Sepharose chromatography of CN[57Co]Cbl-labeled rabbit serum (holo TC II) and the unlabeled serum (apo TC II) showed apo TC II to be more hydrophobic than holo TC II as has been shown for human TC II (Begley et al., Biochem Biophys Res Commun 103:434-441, 1981). Thus mammalian holo TC lis differ in their hydrophobic properties and apo TC II, in man and rabbit, is more hydrophobic than holo TC II. In addition, isolation of the TC II in some animal sera by gel filtration may result in a TC II that is more hydrophobic than the native molecule.