Ermap, a gene coding for a novel erythroid specific adhesion/receptor membrane protein

Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.     

Identity of cells containing apolipoprotein B messenger RNA, in 6- to 12-week postfertilization human embryos

Apolipoprotein B (Apo B) mRNA has been localized by in situ hybridization to various cell types in the liver, gut and yolk sack of the 6- to 12-week postfertilization human conceptus. In the fetal liver it is probable that the immature hepatocytes contain Apo B mRNA. In the yolk sack, the Apo B cDNA probe hybridizes mainly to the large endodermal cells and in the fetal gut the epithelium seems responsible for the majority of Apo B mRNA production. The fetal brain did not show any detectable hybridization to the Apo B probe. Unlike the situation seen in the adult, immunoprecipitation experiments demonstrated that only the B100 form of the protein was synthesized and secreted by the liver, gut and yolk sack at this early stage of human development.     

Developmental changes in the expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers

Cloned cDNAs encoding a number of enzymes involved in cholesterol biosynthesis as well as extracellular and intracellular lipid transport were used to compare the developmental maturation of these biologic functions in the fetal and neonatal rat and human liver. The results of RNA blot hybridization analyses indicate that steady-state levels of rat HMG-CoA synthase, HMG-CoA reductase and prenyl transferase mRNAs are highest in late fetal life and undergo precipitous (up to 80-fold) co-ordinate reductions immediately after parturition. These changes reflect the ability of the fetal rat liver to produce large quantities of cholesterol as well as the repression of this function during the suckling period in response to exogenous dietary cholesterol. Striking co-ordinate patterns of HMG-CoA synthase, reductase and prenyl-transferase mRNA accumulation were also observed in four extrahepatic rat tissues (brain, lung, intestine and kidney) during the perinatal period. The concentrations of all three mRNAs in the 8-week-old human fetal liver are similar to those observed throughout subsequent intrauterine development with less than 2-fold changes noted between the 8th through 25th weeks of gestation. Analysis of the levels of human apo AI, apo AII, apo B and liver fatty acid binding protein mRNAs during this period and in newborn liver specimens also indicated less than 2-3-fold changes. These observations suggest that the 8-week human liver has achieved a high degree of biochemical differentiation with respect to functions involved in lipid metabolism/transport which may be comparable to that present in 19-21 day fetal rat liver. Further analysis of human and rat fetal liver RNAs using cloned cDNAs should permit construction of a developmental time scale correlating hepatic biochemical differentiation to be constructed between these two mammalian species.     

Protein fold analysis of the B30.2-like domain

The B30.2-like domain occurs in some members of a diverse and growing family of proteins containing zinc-binding B-box motifs, whose functions include regulation of cell growth and differentiation. The B30.2-like domain is also found in proteins without the zinc-binding motifs, such as butyrophilin (a transmembrane glycoprotein) and stonustoxin (a secreted cytolytic toxin). Currently, the function for the B30.2-like domain is not clear and the structure of a protein containing this domain has not been solved. The secondary structure prediction methods indicate that the B30.2-like domain consists of fifteen or fewer beta-strands. Fold recognition methods identified different structural topologies for the B30.2-like domains. Secondary structure prediction, deletion and lack of local sequence identity at the C-terminal region for certain members of the family, and packing of known core structures suggest that a structure containing two beta domains is the most probable of these folds. The most C-terminal sequence motif predicted to be a beta-strand in all B30.2-like domains is a potential subdomain boundary based on the sequence-structure alignments. Models of the B30.2-like domains were built based on immunoglobulin-like folds identified by the fold recognition methods to evaluate the possibility of the B30.2 domain adopting known folds and infer putative functional sites. The SPRY domain has been identified as a subdomain within the B30.2-like domain. If the B30.2-like domain is a subclass of the SPRY domain family, then this analysis would suggest that the SPRY domains are members of the immunoglobulin superfamily.     

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.     

Profiles of PrKX expression in developmental mouse embryo and human tissues.

Protein kinase X (PrKX), karyotypically located on the human X chromosome, is a type I cAMP-dependent protein kinase. Although a specific role for PrKX has not yet been defined, PrKX gene expression in mouse and human tissues has been profiled only by in situ hybridization and Northern blot analyses and not by protein expression. To determine more precisely the PrKX protein levels, we developed specific anti-PrKX antibodies and examined gestationally staged mouse embryo sections by immunohistochemistry. These results showed that PrKX is ubiquitously distributed and highly expressed in murine central nervous system and heart tissues in early developmental stages and in most organs at later stages but was not detected in either connective tissues or bone. Using Western blots to detect PrKX, total protein extracts from eight different adult or fetal human tissues including brain, heart, kidney, liver, lung, pancreas, spleen, and thymus were analyzed. Although PrKX protein was present in each of the tissues tested, the protein levels varied depending on tissue type and developmental stage. Very low protein levels were found in heart tissues from a 5-month-old fetus and from an adult, whereas PrKX proteins were more abundant in fetal brain, kidney, and liver tissues compared with adult samples of the same tissue type.     

The carcinoembryonic antigen (CEA) contains multiple immunoglobulin-like domains

The amino acid sequences of human carcinoembryonic antigen deduced from the cDNA sequences have been analysed. This antigen contains seven extracellular domains (previously recognized three highly repetitive domains are further divided into A and B subdomains each) which are strikingly homologous to each other and to immunoglobulin variable regions, poly-Ig receptor and Thy 1.1. The N-terminal domain lacks immunoglobulin-like fold but the other six domains have, suggesting that the CEA belongs to immunoglobulin superfamily.     

Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.

A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Cloning, Tissue Expression Pattern, and Chromosome Location of a Novel Human Gene BRI3BP

A novel cDNA fragment was identified from a human fetal brain cDNA library by using the coding sequence of human BRI3 gene (Accession No. NM015379) as bait in a yeast two-hybrid screening. Then by 5'-RACE (rapid amplification of cDNA end) and electronic hybridization, we obtained a 1.9 kb contig which consists of a novel gene. It was designated as BRI3BP by the HUGO Nomenclature Committee. It contains an open reading frame encoding 251 amino acids. The calculated molecular weight of the deduced protein is 27.8 kU. The predicted isoelectric point is 9.48. Northern hybridization showed its mRNA was highly expressed in brain, kidney, and liver. By RH mapping, the BRI3BP gene was mapped to human chromosome 12q24.2-qter     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Inhibition of mouse SP2/0 myeloma cell growth by the B7-H4 protein vaccine

B7-H4 is a member of B7 family of co-inhibitory molecules and B7-H4 protein is found to be overexpressed in many human cancers and which is usually associated with poor survival. In this study, we developed a therapeutic vaccine made from a fusion protein composed of a tetanus toxoid (TT) T-helper cell epitope and human B7-H4IgV domain (TT-rhB7-H4IgV). We investigated the anti-tumor effect of the TT-rhB7-H4IgV vaccine in BALB/c mice and SP2/0 myeloma growth was significantly suppressed in mice. The TT-rhB7-H4IgV vaccine induced high-titer specific antibodies in mice. Further, the antibodies induced by TT-rhB7-H4IgV vaccine were capable of depleting SP2/0 cells through complement-dependent cytotoxicity (CDC) in vitro. On the other hand, the poor cellular immune response was irrelevant to the therapeutic efficacy. These results indicate that the recombinant TT-rhB7-H4IgV vaccine might be a useful candidate of immunotherapy for the treatment of some tumors associated with abnormal expression of B7-H4. [BMB Reports 2014; 47(7): 399-404]