Antibody-based approach to high-volume genotyping for MIC-1 polymorphism.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution.

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

The propeptide of the transforming growth factor-beta superfamily member, macrophage inhibitory cytokine-1 (MIC-1), is a multifunctional domain that can facilitate protein folding and secretion.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-beta1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major alpha-helix in the mature protein, residues 56-68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-beta1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-beta superfamily protein folding.

Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification of at least five distinct epitope specificities.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.

Expression of a TGF-beta superfamily protein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine. This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism. The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems. Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system. Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase. However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted.

The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1.

Macrophage inhibitory cytokine (MIC-1), a divergent member of the transforming growth factor-beta (TGF-beta) superfamily and activation associated cytokine, is secreted as a 28 kDa dimer. To understand its secretion, we examined its processing in MIC-1-transfected Chinese hamster ovary cells. Mature MIC-1 dimer arises post-endoplasmic reticulum (ER) by proteolytic cleavage of dimeric pro-MIC-1 precursor at a furin-like site. Unlike previously characterized TGF-beta superfamily members, MIC-1 dimers are also secreted in constructs lacking the propeptide. A clue to the function of the propeptide came from the observation that a range of proteasome inhibitors, including lactacystin and MG132, cause major increases in levels of undimerized pro-MIC-1 precursor. There was no effect of proteasome inhibitors on cells expressing mature MIC-1 without the propeptide, suggesting that the propeptide can signal misfolding of MIC-1, leading to proteasomal degradation. Deletion mutagenesis showed the N-terminal 28 amino acids of the propeptide are necessary for proteasomal degradation. This is the first demonstration, to our knowledge, of a quality control function in a propeptide domain of a secretory protein and represents an additional mechanism to ensure correct folding of proteins leaving the ER.

The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women.

Macrophage inhibitory cytokine-1 (MIC-1) is a recently described divergent member of the transforming growth factor-ss superfamily. MIC-1 transcription up-regulation is associated with macrophage activation, and this observation led to its cloning. Northern blots indicate that MIC-1 is also present in human placenta. A sensitive sandwich enzyme-linked immunosorbent assay for the quantification of MIC-1 was developed and used to examine the role of this cytokine in pregnancy. High levels of MIC-1 are present in the sera of pregnant women. The level rises substantially with progress of gestation. MIC-1 can also be detected, in large amounts, in amniotic fluid and placental extracts. In addition, the BeWo placental trophoblastic cell line was found to constitutively express the MIC-1 transcript and secrete large amounts of MIC-1. These findings suggest that the placental trophoblast is a major source of the MIC-1 present in maternal serum and amniotic fluid. We suggest that MIC-1 may promote fetal survival by suppressing the production of maternally derived proinflammatory cytokines within the uterus.

Characterization of human zona pellucida glycoproteins.

The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy.

MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation.

As part of a study to identify novel genes associated with macrophage activation, we have cloned a new member of the transforming growth factor beta (TGF-beta) superfamily designated macrophage inhibitory cytokine 1 (MIC-1). MIC-1 is synthesized as a 62-kDa intracellular protein, which, after cleavage by a furin like protease, is secreted as a 25-kDa disulfide-linked dimeric protein. Sequence analysis indicates that it does not cluster within any existing TGF-beta families, suggesting it may be the first member of a new grouping within the TGF-beta superfamily. Tissue Northern blots show that MIC-1 transcripts are only found abundantly in placenta, although smaller amounts are seen in a limited number of other adult and fetal tissues. MIC-1 is not expressed in resting macrophages but is induced by a number of different activation agents, including phorbol myristate acetate, interleukin 1, tumor necrosis factor alpha, and macrophage colony-stimulating factor but not by lipopolysaccharide or interferon-gamma. We have hypothesized that it may be an autocrine inhibitor of macrophage activation but its major biological role is still uncertain.

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily.

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor beta (TGF-beta) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1beta, tumor necrosis factor alpha (TNF-alpha), interleukin 2, and macrophage colony-stimulating factor but not interferon gamma, or lipopolysaccharide (LPS). Its expression is also increased by TGF-beta. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-alpha production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-beta may serve to limit the later phases of macrophage activation.

IL-3 facilitates lymphocyte hexose transport by enhancing the intrinsic activity of the transport system.

Interleukin 3 (IL-3) promotes the survival and proliferation of hematopoietic cells of various lineages in culture. Like most other hematopoietic colony stimulating factors, its mode of action is unknown. However, binding of the lymphokine induces protein tyrosine phosphorylation and enhanced glucose transport in some myeloid progenitor cells. We have studied the hexose uptake following IL-3 stimulation in IL-3-dependent pro-B cells. IL-3 facilitated the uptake of 2-deoxyglucose within 15 min. Kinetic analysis of the 2-deoxyglucose uptake attributed the enhanced transport to improved transporter function, while other hormones or cytokines affect glucose transport primarily via the number of cell surface transporters.

Redox regulation of a protein tyrosine kinase in the endoplasmic reticulum.

The subcellular localization of the mouse Ltk transmembrane protein tyrosine kinase was studied in transfected COS cells, a mature B lymphocyte line, and a low expressing transfected lymphocyte clone. Indirect immunofluorescence and immunogold staining of COS transfectants and endoglycosidase analysis of both COS transfectants and lymphocytes indicate the unusual localization of Ltk to the endoplasmic reticulum (ER). Ltk resembles a receptor tyrosine kinase; it has a short, glycosylated, and cysteine-rich N-terminal domain. Yet, it appears to function in a ligand-independent mechanism: its in vivo catalytic activity is markedly enhanced by alkylating and thiol-oxidizing agents, and the active fraction of the protein occurs as disulfide-linked multimers. The catalytic activity of Ltk in the ER may be regulated via changes in the cellular redox potential, a novel mechanism for regulating protein tyrosine kinases. The ability to respond to redox changes in the cell may, however, be shared with certain receptor kinases during their passage through the ER.

A molecular genetic linkage map of mouse chromosome 2.

Interspecific backcross mice were used to create a molecular genetic linkage map of chromosome 2. Genomic DNAs from N2 progeny were subjected to Southern blot analysis using molecular probes that identified the Abl, Acra, Ass, C5, Cas-1, Fshb, Gcg, Hox-5.1, Jgf-1, Kras-3, Ltk, Pax-1, Prn-p, and Spna-2 loci; these loci were added to the 11 loci previously mapped to the distal region of chromosome 2 in the same interspecific backcross to generate a composite multilocus linkage map. Several loci mapped near, and may be the same as, known mutations. Comparisons between the mouse and the human genomes indicate that mouse chromosome 2 contains regions homologous to at least six human chromosomes. Mouse models for human diseases are discussed.

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain.

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.

In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA.

A phosphorylated, nonstructural protein of bluetongue virus, NS2, is synthesized throughout the replication cycle in comparatively large amounts. The protein was detected in both the soluble and particulate fraction of the cytoplasm of infected cells. The particulate NS2 could be solubilized in 0.5 M NaCl. It was found that NS2 in the particulate fraction and immunoprecipitates of NS2 from the soluble protein fraction could be phosphorylated in vitro. It is not known whether the kinase involved is of cellular or viral origin, but after purification of NS2 by affinity chromatography on poly(U)-Sepharose it could still by phosphorylated in vitro without the addition of exogenous protein kinase. The affinity of NS2 for nucleic acid was also investigated. The protein was found to bind to single-stranded RNA. In the presence of purified bluetongue virus mRNA, NS2 formed a complex with an estimated S value of about 22S.