The human leucocyte antigen CD48 (MEM-102) is closely related to the activation marker Blast-1

The glycosyl phosphatidylinositol (GPI)-linked antigen recognized by monoclonal antibody (mAb) MEM-102 is expressed on all peripheral blood lymphocytes, both resting and activated. Its properties are very similar to a previously described activation antigen, Blast-1. The amino acid sequence deduced from the structure of cloned cDNA is identical to that of the Blast-1 antigen except for a single amino acid residue. There are several other minor differences in the nucleotide sequence of the Blast-1 and MEM-102 cDNAs that do not affect the predicted structure of the polypeptide product. The amino acid sequence of the first 15 N-terminal residues of the antigen purified from Raji cells is found in the deduced sequence close to the presumed boundary between the leader peptide and mature polypeptide. Properties of the recombinant product expressed in COS cells are similar to the antigen isolated from peripheral blood mononuclear cells (PBMNCs) or B-and T-cells lines. The antigen purified on immobilized mAb MEM-102 is recognized by all six known CD48 mAbs under western blotting conditions. COS cells transfected with MEM-102 cDNA react with all the CD48 mAbs. It is concluded that mAb MEM-102 is directed against the as yet poorly characterized antigen CD48, which is therefore structurally closely related to Blast-1. Several possibilities are discussed that might account for the apparent discrepancy between the broad pan-leucocyte expression of the MEM-102/CD48 antigen and much more restricted expression of the epitope recognized by the previously described mAb defining the Blast-1 antigen.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 37766.     

Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin

We employed cDNA representational difference analysis to identify new genes that are upregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a human hepatoblastoma cell line HepG2. We isolated several TCDD-responsive cDNAs. Sequence analysis revealed that one of them encodes E16/CD98LC/hLAT1, an integral membrane protein involved in multiple cellular functions including cellular transport of L-type amino acids. Northern blot analysis confirmed the TCDD-dependent upregulation of the mRNA. Induction of E16/CD98LC/hLAT1 mRNA by TCDD did not require de novo protein synthesis as revealed by the experiment using cycloheximide. Consistent with the changes at mRNA level, the transport of 3H-leucine into HepG2 cells was significantly increased by TCDD treatment. These findings provide a novel aspect of biological effects of TCDD on human hepatocytes.     

C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.     

TA1/LAT-1/CD98 light chain and system L activity, but not 4F2/CD98 heavy chain, respond to arginine availability in rat hepatic cells. Loss Of response in tumor cells

Tumor associated gene-1/L amino acid transporter-1 (TA1/LAT-1) was recently identified as a light chain of the CD98 amino acid transporter and cellular activation marker. Our previous studies with primary rat hepatocyte cultures demonstrated that TA1 RNA levels were responsive to media amino acid concentrations, suggesting adaptive regulation. High level TA1 expression associated with transformed cells also suggested a role in tumor progression. The present study examined the relationship of TA1/CD98 expression, adaptive response, and associated amino acid transport to neoplastic transformation using a panel of well characterized rat hepatic cell lines. We found 1) increased expression of TA1 in response to amino acid depletion, specific for arginine but not glutamine; 2) loss of TA1 response to arginine in gamma-glutamyl transpeptidase-positive transformed and tumorigenic cells; 3) no appreciable response of 4F2/CD98 heavy chain to arginine levels; and 4) correlation of system L amino acid transport activity in response to arginine with changes in TA1/LAT-1 mRNA but not total immunoreacting protein. Our results suggest this CD98 light chain may act as an environmental sensor, responding to amino acid availability and that its regulation is complex. We hypothesize that altered TA1 expression is an early event in hepatocarcinogenesis giving neoplastic cells a growth or survival advantage, particularly under conditions of limited amino acid availability.     

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.     

The structure and expression of the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2R).

A cDNA clone encoding the receptor for guinea pig immunoglobulin G was isolated from a guinea pig peritoneal macrophage cDNA library. The cloned cDNA encoded 271 amino acids containing an N-terminal signal sequence. The deduced amino acid sequence is most homologous to murine Fc gamma RII beta 2. The receptor protein could be expressed in COS-7 and L cells transfected with the cDNA, suggesting that the expression of this receptor does not require the co-expression of a second chain such as gamma chain of Fc epsilon RI or CD3 zeta chain. The transformant L cells showed the binding to both the guinea pig IgG1 and IgG2 antibodies complexed with antigen, indicating that the cDNA we cloned was the one for guinea pig Fc gamma 1/gamma 2R.     

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.     

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.     

Analysis of a monoclonal rat antibody directed to the alpha-chain variable region (V alpha 3) of the mouse T cell antigen receptor

Three rat mAb, RR3-15, RR3-16, and RR3-18, were established by fusing spleen cells from a rat immunized with the male Ag-specific cytolytic T cell clone, OH6, to mouse myeloma cells. The mAb was identified by their capacity to focus the cytolytic activity of the OH6 CTL clone on nonspecific target cells via FcR-FcR interaction. That all three mAb recognized the OH6 TCR was confirmed by immunoprecipitation studies in which each antibody precipitated a 90 kDa disulfide-linked heterodimer characteristic of the TCR. Surface immunofluorescence staining of a panel of T cell lines and splenic T cell populations showed that RR3-16 reacted not only to the OH6 T cell clone but also to a minor fraction of normal T cells. This reactivity was found to be due to the expression of a gene in the V alpha 3 family. However, RR3-16 did not react with all T cell lines and clones known to express genes from the V alpha 3 family. cDNA sequences of three independent RR3-16+ T cell hybridomas analyzed by polymerase chain reaction were identical to the previously published V alpha 3 sequence of the CTL clone C9. Thus, the mAb RR3-16 is specific for a single member of the TCR V alpha 3 gene family. Analysis of the expression of RR3-16+ TCR in CD4+ and CD8+ subsets of peripheral T cells demonstrated preferential expression on CD8+ T cells, suggesting regulated expression of this particular TCR V alpha gene.     

Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes

Here we characterize the properties and expression pattern of Siglec-9 (sialic acid-binding Ig-like lectin-9), a new member of the Siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding Siglec-9 was isolated from a dibutyryl cAMP-treated HL-60 cell cDNA library. Siglec-9 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two putative tyrosine-based signaling motifs. Overall, Siglec-9 is approximately 80% identical in amino acid sequence to Siglec-7, suggesting that the genes encoding these two proteins arose relatively recently by gene duplication. Binding assays showed that, similar to Siglec-7, Siglec-9 recognized sialic acid in either the alpha2,3- or alpha2, 6-glycosidic linkage to galactose. Using a specific mAb, Siglec-9 was found to be expressed at high or intermediate levels by monocytes, neutrophils, and a minor population of CD16(+), CD56(-) cells. Weaker expression was observed on approximately 50% of B cells and NK cells and minor subsets of CD8(+) T cells and CD4(+) T cells. These results show that despite their high degree of sequence similarity, Siglec-7 and Siglec-9 have distinct expression profiles.     

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642-1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.     

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.     

The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.

Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Microtubule-associated proteins 1A and LC2. Two proteins encoded in one messenger RNA.

The deduced amino acid sequence for the filamentous microtubule-associated protein (MAP) 1A, thought to be involved in stabilizing the mature neuronal cytoskeleton, has been determined from a series of overlapping cDNA clones. Though previously described as biochemically and immunologically distinct from MAP1B, we now demonstrate that MAP1A is structurally related to MAP1B, a protein associated with neurite outgrowth and process plasticity. The two MAPs exhibit regional amino acid sequence similarities spanning their potential microtubule binding domains placing both into a new MAP family. The cDNA sequence encoding MAP1A was also found to encode one of its associated light chains (LC) called LC2. Both proteins are found on a single mRNA in the same open reading frame and are translated as a pre-MAP1A/LC2-protein. The topological relationship between MAP1A and LC2 coding sequences is, therefore, identical to that previously shown for MAP1B and LC1 (Hammarback, J. A., Obar, R. A., Hughes, S. M., and Vallee, R. B. (1991) Neuron 7, 129-139). Based on these and earlier results, we conclude that LC1 and LC2 are structurally related polypeptides generated from distinct MAP polyprotein precursors but free to exchange between the two MAPs.     

Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes--a molecule related to nerve growth factor receptor.

The antigen recognized by the monoclonal antibody (mAb) MRC OX40 is present on activated rat CD4 positive T lymphocytes but not other cells. cDNA clones were isolated from an expression library using the MRC OX40 mAb and the protein sequence for the OX40 antigen deduced. It contains a typical signal sequence and a single putative transmembrane sequence of 25 predominantly hydrophobic amino acids giving an extracellular domain of 191 amino acids and a cytoplasmic domain of 36 amino acids. The sequence of the extracellular domain includes a cysteine-rich region with sequence similarities with the low affinity nerve growth factor receptor (NGFR) of neurons and the CD40 antigen present on human B cells. Within this region three cysteine-rich motifs can be recognized in OX40 compared with four similar motifs in both NGFR and CD40. OX40, CD40 and NGFR constitute a new superfamily of molecules with expression including lymphoid cells (OX40, CD40) and neuronal cells (NGFR). This is reminiscent of the immunoglobulin superfamily whose molecules are variously found at the surface of lymphoid or brain cells or both.     

Molecular cloning,expression pattern and chromosomal mapping of pig CD69

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.     

The second EF-hand is responsible for the isoform-specific sorting of myosin essential light chain.

It has been known that isoforms of myosin essential light chain (LC) exhibit the isoform-specific sorting within cardiac myocytes and fibroblasts. In order to analyze which domain of LC is responsible for the sorting, various chimeric cDNA constructs between human nonmuscle isoform (LC3nm) and chicken fast skeletal muscle isoform (LC3f) were generated and expressed in cultured chicken cardiac myocytes. If chimeras contained LC3f sequence at the place that was restricted by BssHII and PstI, they were preferentially sorted to sarcomeres and precisely localized at A-bands, and their incorporation levels into the A-bands were identical with that of the wild type LC3f. However, other chimeras were distributed throughout the cytoplasm like the wild type LC3nm. Comparison of amino acid sequences revealed that 12 amino acids are different between chicken LC3f and human LC3nm in the BssHII-PstI fragment, and these amino acids are located within the second EF-hand of LC. These results indicated that the second EF-hand is responsible for the isoform-specific sorting of LC. Although the second EF-hand is not included in the key contacts with myosin heavy chain, it is supposed that this domain is important for the relative disposition of neighboring domains. Thus, the 12 amino acids in the second EF-hand might play a key role for modulation of overall configuration of LC, thereby influencing the precise association of the key contacts.