A human homolog of the mouse CD8 molecule,Lyt-3: genomic sequence and expression

The CD8 antigen is a marker for those cytotoxic T cells that recognize antigen in the context of class I major histocompatibility antigens (MHC) and has now been identified in many species. In rodents the CD8 antigen is a heterodimer of two distinct chains, Lyt-2 and Lyt-3 in the mouse and OX-8 M _r 32 000 and 37 000 chains in the rat. Human CD8 has consistently been described as a homodimer/homomultimer on mature T cells made up of one chain homologous to the Lyt-2 and OX-8 M _r 32 000 chains. This paper identifies a human equivalent of the second rodent CD8 chain (Lyt-3 and OX-8 M _r 37 000 chains) at the genomic level and shows that this gene is transcribed in human thymocytes and in some acute leukemic T-cell lines. The existence of a human Lyt-3 homolog raises the possibility that human CD8, like mouse CD8, may exist as a heterodimer.     

Isolation and characterization of mouse CD7 cDNA

The human CD7 antigen is a glycoprotein, M _r 40 000, expressed on the surface of peripheral blood T-lymphocytes and thymocytes, and is the earliest surface antigen to appear on T-cell lineage cells. In this study, putative mouse CD7 cDNA was identified based on its similarities with human CD7. Five independent clones originating from the same mRNA species were isolated (designated as mCD7) by screening a mouse thymocyte cDNA library with human CD7 cDNA, J61, under moderate stringency. The longest insert of a 995 base pair had an open reading frame of 210 amino acids. Northern blot analysis using the mouse CD7 cDNA probe demonstrated a single 1.2 kilobase mRNA ni the thymus, spleen, bone marrow, and small intestine. The protein deduced from mCD7 cDNA consisted of the leader, extracellular, transmembrane, and cytoplasmic domains of 24, 126, 21, and 39 amino acids, respectively, based on the hydrophobicity plot and the structure of human CD7. The extracellualr domain contained three potential N-glycosilation sites, while the cytoplasmic domain contained one potential protein kinase C phosphorylation site. The amino acid sequence had 45.5% similarity with human CD7, while the similarities for the individual domains ranged from 49.2% to 63.2%. The six highly conserved regions, which may possibly be involved with still unknown CD7-mediated functions, were located in the extracellular and cytoplasmic domains.The nucleotide sequence data reported in this paper have seen submitted to the GenBank, DDBJ, and EMBL nucleotide sequence database and have been assigned the accession number D10329.     

Human non-lineage antigen,CD46 (HuLy-m5): purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins

CD46, until recently known as HuLy-m5, is a non-lineage restricted surface antigen ubiquitously expressed by almost all human cells except erythrocytes. The CD46 antigen is identified by the E4.3 monoclonal antibody (mAb) and exists at the surface of human peripheral blood lymphocytes (PBLs) as two acidic, non-disulfide bonded chains, and , ofM _r 66 000 and 56 000. Receptor density analysis showed that CD46 was of moderately low abundance on PBLs with 7.5×10³ molecules present on each cell. The two chains of CD46 were purified (144 000-fold) by immunoaffinity-chromatography with E4.3 mAb from the plasma membranes of a human spleen infiltrated with chronic myelogenous leukemia cells. Amino acid sequence analysis of the NH₂-terminal of both and chains yielded the same sequence; XEEPPQ/TFEAMELIGKPKPYYEIGE. Peptide mapping studies confirmed that both CD46 chains were closely related, except for one peptide fragment. This amino acid sequence is identical to that of the NH₂-terminal of the recently cloned membrane co-factor protein (MCP), a membrane protein that binds the C3b and C4b fragments of complement and acts as a co-factor for I protein-mediated decay of the complement convertases. CD46 shares a cross-reactive epitope with some primate retroviruses, and this may indicate that some retroviruses mimic the mechanisms used by autologous human cells to evade complement-mediated immune clearance. Offprint requests to: I. F. C. McKenzie.     

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH₂-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH₂-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X₃-Cys-X₃-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.     

Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.     

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins

Summary The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH₂-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH₂-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis.Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH₂-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.     

The isolation of cDNA clones for CD48

HuLy-m3 is an M _r 47 000 pan-leukocyte antigen detected by the monoclonal antibody (mAb) 5-4.8. This report describes the isolation and analysis of a cDNA clone encoding HuLy-m3. Serological analysis demonstrated that antibodies of the CD48 cluster also reacted with transfected cells expressing HuLy-m3. The DNA sequence of the clone suggests linkage to the cell membrane through a glycosyl phosphatidylinositol tail and this was verified experimentally. Sequence similarity with the human B-cell activation antigen Blast-1 was noted.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 59904.     

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene

 The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned. Received: 22 June 1998 / Revised: 16 September 1998     

Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes.

The MRC OX-8 antigen is a marker of the rat cytotoxic T lymphocytes that consists of disulphide-linked chains of mol. wts. 37 and 32 kd. It is thought to be equivalent to the human T8 and mouse Lyt2,3 antigens (all now called CD8 antigens). MRC OX-8 antigen was purified from thymocytes using a monoclonal antibody column and because antigenicity was retained after reduction and alkylation the two polypeptide chains could be separated by a subsequent affinity chromatography step. Peptides were isolated from each chain and their sequences determined. A cDNA probe coding for the mouse CD8 antigen (pLY2C-1 provided by Dr L. A. Herzenberg) was used to obtain rat cDNA clones from which the sequence of the equivalent rat molecule was determined. Peptides from the 32-kd chain were identified in this translated sequence whereas peptides from the 37-kd chain were not. The 32-kd polypeptide sequence consisted of 210 amino acids and had one possible N-linked glycosylation site. The N-terminal part of the sequence was surprisingly different from both its mouse and human counterparts but, as in the other two species, it showed a clear relationship to Ig V domains.     

The genes for CD37, CD53, and R2, all members of a novel gene family,are located on different chromosomes

CD37, CD53, and R2 leukocyte surface antigens are members of a novel family of structurally related proteins. They all have four transmembrane-spanning domains with a single major extracellular loop. The CD37 is expressed on B cells and on a sub-population of T cells. The CD53 is known as a panleukocyte marker. The R2 protein is an activation antigen of T cells. The CD37, CD53, and R2 genes were assigned with the help of human/rodent somatic cell hybrids and human-specific probes to human chromosomes 19, 1, and 11, respectively. For the regional assignment, various deletion hybrids were used to map CD37 to 19p13-q13.4, CD53 to 1p12-p31, and R2 to 11p12.     

MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains.     

Characterization of the human homolog of the rat MRC OX-2 membrane glycoprotein

The MRC OX-2 antigen is a membrane glycoprotein present on rat thymocytes, neurons, follicular dendritic cells, endothelium, and some smooth muscle. The sequence of 248 amino acids has similarities to Ig domains organized with one V-like domain, one C-like domain, and transmembrane and cytoplasmic regions. Thus it resembles a T-cell receptor chain but shows no sequence divergence. We report the characterization of the human gene for this molecule. Its exon organization is similar to that found for immunoglobulins although the region with similarities to Ig J regions is found within the same exon as the V-like domain. Human MRC OX-2 is expressed at the mRNA level in brain and B-cell lines but not detected in liver or T-cell lines. It does not obviously correspond to any previously defined leukocyte antigen. The sequence homology for the human and rat MRC OX-2 molecules is higher for the Ig-related region (75 %) than for many other Ig-related molecules and very high in the transmembrane region (96 %), implying a functional role other than simply its anchoring into the membrane.     

A Gene Encoding a Cytochrome c Oxidase-Like Protein Is Located Closely to the Cytochrome c-553 Gene in the Anaerobic Bacterium,Desulfovibrio vulgaris (Miyazaki F)

The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.     

Molecular Characterization of Genes Encoding a Novel ABC Transporter in Thermoanaerobacterium thermosulfurigenes EM1

The nucleotide sequence of two open reading frames (ORFs) from Thermoanaerobacterium thermosulfurigenes EM1 was determined that encode proteins with similarity to components of ATP-binding cassette (ABC) transport systems. Sequence analysis suggests that the deduced proteins AbcA and AbcB consist of an NH₂-terminal membrane-spanning domain and a COOH-terminal ATP-binding domain. The deduced proteins AbcA and AbcB showed highest similarity to proteins of the MsbA subfamily of ABC transporters. AbcA and AbcB probably function as a heterodimer. An ORF predicted to encode the primary sigma factor SigA was identified downstream of abcB. Received: 11 March 1997 / Accepted: 14 April 1997     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase