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.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.     

Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily

The CD19 (B4) molecule is a m.w. 95,000 cell-surface protein of human B lymphocytes that is expressed before Ig and persists throughout differentiation. In this report, cDNA clones that encode the CD19 molecule were isolated and the amino acid sequence of CD19 was determined. A cDNA clone that selectively hybridized to RNA from CD19+ cell lines was selected from a human tonsilar cDNA library using differential hybridization. This cDNA was used to isolate additional cDNA clones. Four of the five longest cDNA clones isolated were sequenced and found to contain unique sequences presumed to be introns. One clone, pB4-19, was near full length (2.1 kb) and did not contain these putative introns. pB4-19 contained an 1685 bp open reading frame that could encode a protein of about 60 kDa. COS cells that were transfected with pB4-19 expressed a nascent cell surface structure reactive with the anti-B4 antibody. Immunoprecipitation of this structure from surface-iodinated COS cells with the anti-B4 antibody revealed a m.w. 85,000 protein. Northern blot analysis indicated that pB4-19 hybridized with a predominant mRNA species of 2.4 kb and a minor species of 1.5 kb, found in only CD19+ cells. The pre-B cell line, PB-697, also expressed four larger RNA species that hybridized with pB4-19. cDNA clones that encode the putative cytoplasmic portion (247 amino acids) of the mouse CD19 molecule were also isolated and found to be highly homologous (79 and 75%) with the human CD19 nucleotide and amino acid sequences. The deduced amino acid sequence of the CD19 cytoplasmic tail shared no significant homology with other known proteins but the putative extracellular region contained two Ig-like domains indicating that CD19 is a new member of the Ig superfamily.     

The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with O-linked glycosylation of one third of its extracellular amino acids.

Leukosialin is one of the major glycoproteins of thymocytes and T lymphocytes and is notable for a very high content of O-linked carbohydrate structures. The full protein sequence for rat leukosialin as translated from cDNA clones is now reported. The molecule contains 371 amino acids with 224 residues outside the cell, one transmembrane sequence and 124 cytoplasmic residues. Data from the peptide sequence and carbohydrate composition suggest that one in three of the extracellular amino acids may be O-glycosylated with no N-linked glycosylation sites. The cDNA sequence contained a CpG rich region in the 3' coding sequence and a large 3' non-coding region which included tandem repeats of the sequence GGAT.     

Location of a threonine residue in the alpha-subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel.

By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the alpha-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (alpha T264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two alpha-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel.     

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

A role in transmembrane signaling for the cytoplasmic domain of the CD2 T lymphocyte surface antigen

The CD2 antigen can mediate mitogenesis of T lymphocytes after binding combinations of monoclonal antibodies. To examine the importance of the cytoplasmic domain in signaling, rat CD2 cDNA has been transfected into the human Jurkat cell line and triggering of an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i) has been assayed. In cells expressing full-length CD2, a clear signal was triggered with anti-CD2 monoclonal antibodies. In contrast, a barely detectable increase in [Ca2+]i occurred with mutant rat CD2 molecules that included only 6 or 40 amino acids of the full-length cytoplasmic domain of 116 residues. It thus appears that the CD2 cytoplasmic domain plays a role in the signaling event.     

Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain.

The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals.     

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules

 CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice. Received: 1 July 1998 / Revised: 31 August 1998     

Molecular cloning of rat cytolysin

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.     

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2, respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numerous structural similarities.