Cyclic activity of the receptors of cobalamin bound to transcobalamin II

The activity of receptors specific for human transcobalamin II-Cobalamin (TC II-Cbl) were measured in virus-transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibro lasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor number and not by changes in affinity between receptors and TC II-Cbl. The basis of the change in fibroblasts was not clear. The Cbl-dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II-Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II-Cbl for the regulation of the entry of Cbl into cells.     

The function of cellular transcobalamin II in cultured human cells

The known function of human transcobalamin II (TC II) is to transport cobalamin (Cbl) in the circulation to tissue receptors for TC II-Cbl. Several types of human cells synthesize apo (unsaturated) TC II and the present study was conducted in order to evaluate possible functions of this endogenous TC II. The approach consisted of a correlation between the abilities of cultured cells to produce apo TC II and to internalized Cbl when presented in the free form. The amount of apo TC II produced by six lines of cultured human cells ranged from abundant to nil. The amount of free Cbl internalized by these cells correlated directly with the capacity to produce apo TC II. The interactions between endogenous TC II and free Cbl took place either at the cell surface or in the medium surrounding the cell. It was also shown that cells in culture contain free Cbl and release free Cbl into the surrounding medium. Thus it was concluded that the apo TC II produced by human cells remains intact to interact with free Cbl and to participate in the cellular metabolism of Cbl.     

The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma)

The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way.     

Methionine dependency of cultured human lymphocytes

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.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met ^? Hcy ⁺ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met ^? Hcy ⁺ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met ^? Hcy⁺ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-pindependent human fibroblasts when incubated in Met ^? Hcy⁺ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met ^? Hcy⁺ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.     

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.     

Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.     

Cbl-mediated ubiquitinylation and negative regulation of Vav

The Cbl ubiquitin ligase has emerged as a negative regulator of receptor and non-receptor tyrosine kinases. Cbl is known to associate with the proto-oncogene product Vav, a hematopoietic-restricted Rac guanine nucleotide exchange factor, but the consequences of this interaction remain to be elucidated. Using immortalized T cell lines from Cbl(+/+) and Cbl(-/-) mice, and transfection analyses in 293T cells, we demonstrate that Vav undergoes Cbl-dependent ubiquitinylation under conditions that promote Cbl and Vav phosphorylation. Interaction with Cbl also induced the loss of phosphorylated Vav. In addition, we show that an activated Vav mutant (Vav-Y174F) is more sensitive to Cbl-dependent ubiquitinylation. We demonstrate that the Cbl-dependent ubiquitinylation of Vav requires Cbl/Vav association through phosphorylated Tyr-700 on Cbl, and also requires an intact Cbl RING finger domain. Finally, using transfection analyses in the Jurkat T cell line, we show that Cbl, but not its ubiquitin ligase mutant, can inhibit Vav-dependent signaling. Thus, our findings strongly support the role of Cbl, via its ubiquitin ligase activity, as a negative regulator of activated Vav.     

Effect of Cobalamin Deficiency on the Biosynthesis of Phosphatidylcholine in Euglena gracilis

Euglena gracilis requires cobalamin (Cbl) as an essential growth factor. Phosphatidylcholine (PC) synthesis was greatly reduced by Cbl deficiency. Rapid cell division occurred after Cbl was replenished, and PC was actively synthesized during the cell divisions. When the deficient cells were given methionine (a precursor for the choline moiety), active synthesis of PC occurred even without the Cbl supplement, although cell division was not induced. As methionine synthase in Euglena requires methylcobalamin as a coenzyme, decrease in methionine synthesis may account for reduced PC synthesis under Cbl-deficient conditions. Phosphatidyleth-anolamine and phosphatidylserine synthesis were also suppressed, commensurate with decrease of PC synthesis, under Cbl deficiency, even though Cbl is not thought to participate in their synthesis. In contrast, a lot of triglyceride and wax ester accumulated in Cbl-deficient cells. Moreover, Cbl depletion altered fatty acid composition, notably due to increased proportion of odd-numbered fatty acids     

Cobalamin metabolism in cultured human chorionic villus cells

Cobalamin (Cbl, vitamin B₁₂) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[⁵⁷Co]Cbl) complexed to TCII. This internalized CN[⁵⁷Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [¹⁴C]CH₃-tetrahydrofolate and [¹⁴C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley-Liss, Inc.     

Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells

The bacterial pathogen Listeria monocytogenes uses the surface protein InlB to invade a variety of cell types. The interaction of InlB with the hepatocyte growth-factor receptor, Met, is crucial for infection to occur. Remarkably, the ubiquitin ligase Cbl is rapidly recruited to InlB-activated Met. Recent studies have shown that ligand-dependent endocytosis of Met and other receptor tyrosine kinases is triggered by monoubiquitination of the receptor, a process that is mediated by Cbl. Here, we show that purified InlB induces the Cbl-dependent monoubiquitination and endocytosis of Met. We then demonstrate that the bacterium exploits the ubiquitin-dependent endocytosis machinery to invade mammalian cells. First, we show that L. monocytogenes colocalizes with Met, EEA1, Cbl, clathrin and dynamin during entry. Then, we assess the role of different proteins of the endocytic machinery during L. monocytogenes infection. Over-expression or down-regulation of Cbl, respectively, increases or decreases bacterial invasion. Furthermore, RNA interference-mediated knock-down of major components of the endocytic machinery (for example, clathrin, dynamin, eps15, Grb2, CIN85, CD2AP, cortactin and Hrs), inhibit bacterial entry, establishing that the endocytic machinery is key to the bacterial internalization process.     

Congenital disorders of vitamin B12 transport and their contributions to concepts. II.

Congenital deficiencies of Transcobalamin II (TC II) and R binders of vitamin B12 (B12, cobalamin, Cbl) have been described in several families. The deficiency of TC II exists as at least three variants. The deficiency of TC II is expressed by a profound megaloblastic pancytopenia during the first few weeks of life, but the serum Cbl is normal. In contrast, the deficiency of R binder is asymptomatic, tissues are replete in Cbl, but the serum Cbl is low. All of the R binder in the several body sources is under the same genetic control. Studies of the congenital deficiency TC II suggest the following: (1) The function of TC II is the promotion of cell uptake of physiologic amounts of Cbl, which can also be accomplished by very large amounts of Cbl, and not in any intracellular process. (2) TC II is essential for the absorption, postabsorptive distribution, and recycling of TC II. (3) The metabolic consequences of TC II deficiency are expressed primarily in rapidly dividing cells probably because they are dependent upon the constant need for new Cbl.     

The metabolic defect of methionine dependence occurs frequently in human tumor cell lines

Methionine dependence is the inability of cells to grow when methionine (Met) is replaced by its immediate precursor homocysteine (Hcy) in the culture medium (Met^?Hcy⁺ medium). All normal unestablished cell strains tested to date have been shown to be methionine-independent and thus grow almost as well in Met^?Hcy⁺ medium as they do in Met⁺Hcy^? medium. Results presented here indicate that out of 23 cell lines derived from diverse types of human tumors, 11 do not grow at all in Met^?Hcy⁺ medium and are absolutely methionine-dependent and 3 grow only slightly in this medium. Many of the tumor cell lines tested have little else in common other than the fact that they are methionine-dependent. The high frequency of occurrence of methionine dependence in diverse types of human tumor cells indicates that methionine dependence may be an important aspect of oncogenic transformation and therapeutically exploitable.     

Brain energy metabolism is compromised by the metabolites accumulating in homocystinuria

Homocystinuria is an inborn error of metabolism caused by severe deficiency of cystathionine beta-synthase activity. It is biochemically characterized by tissue accumulation of homocysteine (Hcy) and methionine (Met). Homocystinuric patients present a variable degree of neurological dysfunction whose pathophysiology is poorly understood. In the present study, we investigated the in vitro effect of Hcy and Met on some parameters of energy metabolism in hippocampus of rats. CO(2) production from [U-14C] acetate, glucose uptake and lactate release were assessed by incubating hippocampus prisms from 28-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of Hcy (10-500 microM) or Met (0.2-2.0mM). Hcy and Met decreased CO(2) production in a dose-dependent manner and increased lactate release. In contrast, glucose uptake was not altered by the metabolites. The effect of Hcy and Met on cytochrome c oxidase activity was also studied. It was observed that Met did not alter this enzyme activity, in contrast with Hcy, which significantly inhibited cytochrome c oxidase activity. It is suggested that impairment of brain energy metabolism caused by the metabolites accumulating in homocystinuria may be related to the neurological symptoms present in homocystinuric patients.     

Metabolism to methionine and growth stimulation by 5'-methylthioadenosine and 5'-methylthioinosine in mammalian cells

Viable human and murine lymphoblasts, and normal human tissue extracts, converted the thioether nucleosides 5'-methylthioadenosine (MeSAdo) and 5'-methylthioinosine (MeSIno) to methionine. Both MeSAdo and MeSIno, but not homocysteine, supported the short-term growth of human or murine lymphoblasts in methionine deficient medium. However, MeSAdo at concentrations greater than 25 microM inhibited cell growth. MeSIno was non-toxic at concentrations up to 200 microM, and supported the long-term growth of lymphoblasts in methionine-free medium.