The availability of therapeutic hydroxocobalamin to cells

Hydroxocobalamin (OH-Cbl), when used to treat vitamin B12 deficiency, is better retained by the body than is cyanocobalamin (CN-Cbl), but the availability to cells has not been studied systematically. In a series of experiments, we compared the uptake and internalization of OH-Cbl and CN-Cbl bound to transcobalamin II (TCII) by a human cell model, the HeLa cell. TCII-OH-Cbl was: (1) taken up in larger amounts per unit time, (2) the greater uptake was not a consequence of more effective attachment to receptors of TCII-Cbl nor to a more rapid regeneration of receptors, (3) the difference was expressed during the phase of internalization of TCII-Cbl, (4) with CN-Cbl, the stages of binding to receptors plus internalization were more readily reversed, and (5) larger amounts of OH-Cbl were internalized and converted to active coenzyme forms of Cbl. When injected into a healthy person, 200 micrograms of OH-Cbl was better retained in the circulation than 200 micrograms of CN-Cbl. When added in vitro in equivalent amounts, more OH-Cbl was bound to nonspecific plasma proteins. This greater and broader binding neither enhanced nor interfered with the uptake of Cbl by cells, which was determined by the amount of Cbl bound physiologically to TCII. It was concluded that OH-Cbl is a more efficient form of treatment of the common types of Cbl deficiency, principally because of the better retention, which requires less frequent injections, but also because of greater availability to cells.     

The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.     

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.     

Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid.

A component of soluble terminal complement complexes was identified and affinity-purified to homogeneity by using a monoclonal antibody previously developed against the soluble C5b-9 complex. The protein, which we have designated complement cytolysis inhibitor (CLI), has a molecular mass of 70 kDa and consists of two nonidentical, disulfide-linked subunits of 35 kDa. Partial amino acid sequences determined for the amino-termini of the two subunits were identical with those of a recently characterized serum protein called SP-40,40. An almost full-length cDNA clone of 1651 base pairs was isolated from a human liver cDNA library by using long synthetic oligonucleotides as probes. The encoded amino acid sequence of CLI consists of 427 amino acid residues preceded by a 21-residue-long typical signal peptide and shows an overall 75.6% amino acid sequence homology to sulfated glycoprotein 2 (SGP-2), a major Sertoli cell-derived protein of rat testis fluid. As in SGP-2, proteolytic processing between residues 206 and 207 yields the two disulfide-linked subunits of plasma CLI. CLI and SGP-2 were shown to be orthologous single-copy genes in humans and rats by Southern blotting experiments. In addition, CLI was immunologically identified in human seminal plasma. Functional studies with purified terminal complement components showed that CLI suppresses the cytolytic potential of nascent C5b-7 complexes at physiological blood plasma concentrations (approximately 50 micrograms/ml). Its presence on the surface of mature sperm cells and its relative abundance in seminal plasma (approximately 250 micrograms/ml) suggest that CLI protects sperm cells and epithelial tissues against complement attack in the male reproductive tract.     

Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.     

Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.     

Structure and expression of human dihydropteridine reductase.

Dihydropteridine reductase (DHPR; EC 1.6.99.7) catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterin-dependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector lambda gt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR.     

Characterization of a cDNA coding for human factor X.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human factor X, a plasma protein participating in the middle phase of the blood coagulation cascade. Ten positive clones were isolated from 2 X 10(6) phage and plaque purified. The cDNA in the phage containing the largest insert has been sequenced and shown to code for human factor X. This cDNA insert contained 1137 base pairs coding for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a short 3' noncoding region, and a poly(A) tail. The sequence of A-T-T-A-A-A, which functions as a potential recognition site for polyadenylylation or processing, was present in the 3' end of the coding sequence and preceded the stop codon of TGA by 1 base pair and the poly(A) tail by 14 base pairs. The amino acid sequence deduced from the cDNA indicated that factor X is synthesized as a single-chain polypeptide containing the light and heavy chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the light and heavy chains by cleavage of two (or more) internal peptide bonds. In plasma, these two chains are linked together by a disulfide bond. The DNA sequence coding for the active site of human factor X showed a high degree of identity with prothrombin and factor IX, two other vitamin K-dependent serine proteases that participate in blood coagulation. These data along with the protein sequence data previously published for the light chain of human factor X establish the complete amino acid sequence for the mature protein present in plasma.     

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.     

Transplacental Transport in the Rabbit of Vitamin B12 Bound to Human Transcobalamins I, II and III

Transcobalamins I, II and III (TCI, TCII, TCIII) were purified from human serum, saturated with 57Co-vitamin B12 (57Co-B12), and injected into pregnant rabbits. Whole body retention of the 57Co-B12 averaged 91% and was similar for each of the three vitamin B12 binders. A maximum of 62% of the injected 57Co-B12 was found in fetal tissues when the vitamin was injected bound to TCII, but only 6--7% when bound to TCI or TCIII. Thus TCII is responsible for the delivery of vitamin B12 to the fetus.     

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.     

Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identical to p36, a protein associated to lupus inclusion formation in response to alpha-interferon

Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)     

Transcobalamin II deficiency with methylmalonic aciduria in three sisters

Transcobalamin II (TC II) is a plasma protein that binds vitamin B₁₂ (cobalamin, Cbl) and facilitates cellular Cbl uptake by receptor-mediated endocytosis. In autosomal recessive TC II deficiency, intracellular Cbl deficiency results in an early onset of megaloblastic anaemia that may be accompanied by neurological abnormalities. Inadequate treatment may lead to neurological abnormalities. We describe three sisters, the daughters of first cousins of Moroccan origin, with TC II deficiency requiring continuous and long-term vitamin B₁₂ treatment. The diagnosis was suspected from the finding of low unsaturated vitamin B₁₂ binding capacity and confirmed by absence of detectable TC II by radioimmunoassay and by inability of cultured fibroblasts to synthesize TC II.     

Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae.

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.     

Carboxyl-terminal amino acid sequences of two variant forms of the gamma chain of human plasma fibrinogen.

The gamma chain of human fibrinogen has been shown to be heterogeneous; three forms of various sizes are normally present in plasma. To further characterize the two less prevalent elongated variants, we have purified the three forms of the gamma chain, isolated unique tryptic fragments, and sequenced the variant portions of each chain. The intermediate-sized form (gamma 55) has a sequence identical to the longest variant (gamma 57.5) from residue Val-408 to Pro-423, which is the C terminus of the gamma 55 chain. The gamma 57.5 chain extends an additional four amino acids. The C-terminal amino acid sequence and corresponding nucleotide sequence of the gamma 55 chain show marked similarity with an elongated gamma-chain variant of rat fibrinogen. In addition, a remarkable similarity in both amino acid and nucleotide sequence was noted between the human gamma 55 chain and the C-terminal extension in human and bovine A alpha chain of fibrinogen, which are encoded by the cDNA but are posttranslationally cleaved and are not found in plasma. The findings suggest an evolutionary conservation in the C-terminal amino acid sequence found in both the fibrinogen A alpha and gamma chains in several species.