Mark D. Wright

The ins and outs of the transmembrane 4 superfamily

The recently discovered transmembrane 4 superfamily comprises a group of cell-surface proteins that are characterized by the presence of four hydrophobic domains, which are presumed to be membrane spanning. At least seven of these molecules are expressed on leukocytes, and it seems likely that they mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility.

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.

Molecular and serological characteristics of the glutathione S‐transferases of Schistosoma japonicum and Schistosoma mansoni

Summary When aqueous extracts of Schistosoma japonicum and S. mansoni adult worms are passed over columns of glutathione‐conjugated agarose, two molecular species of Mr 26000 and Mr 28000 are detected in eluates as analysed by SDS‐PAGE, these eluates having glutathione S‐transferase (GST) activity. The molecules, termed Sj26 and Sj28 from S. japonicum and Sm26 and Sm28 from S. mansoni, can be immunogenic in rabbits or mice and appear not to be linked together as subunits of GST heterodimers. The elution profile of SjGST (Sj26 + Sj28) from glutathione columns resembles that of SmGST (Sm26 + Sm28) and, by peptide mapping, radioiodinated Sj26 and Sm26 are related as are the two Mr28000 molecules. Similarities between radioiodinated Sj2S and Sm28 are also obvious on two‐dimensional gel electrophoresis with some differences being observed between Sj26 and Sm26. The Mr28000 molecules are more prominent than the Mr 26000 molecules and, although Sj28 is a poor immunogen in mice, immunological cross‐reactivity between Sj28 and Sm28 is generally more readily detected than that between Sj26 and Sm26. Whether experimental vaccination against schistosomiasis japonica and schistosomiasis mansoni reported with cloned GSTs can be improved by incorporation of both Mr28000 and Mr 26000 species into the vaccine remains to be determined. On this point, the present data suggest that vaccination of mice with Sj26 plus Sm28 should be a useful means of increasing antibody responses to the GSTs of S. japonicum.

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.

The functional and immunological significance of some schistosome surface molecules

The molecules discussed in this review include some of the leading vaccine candidates in schistosomiasis: the glutathione S-transferases, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase and the 23 and 25 kDa surface integral membrane proteins. Mark Wright, Kathy Davem and Graham Mitchell highlight the possible biological roles and immunological relevance of these molecules.

The use of intracellular single-chain antibody fragments to inhibit specifically the expression of cell surface molecules.

One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly.

CD2 physically associates with CD5 in rat T lymphocytes with the involvement of both extracellular and intracellular domains

T lymphocytes can be activated and induced to proliferate through stimulation of the CD2 glycoprotein with functional combinations of CD2 antibodies. However, this mechanism of signal transduction via CD2 is still not fully understood. We have investigated which molecules on the T cell surface preferentially associate in Cis with CD2 and may regulate its signaling properties. Though a quantification method we found that CD5 represents the antigen capable of co‐precipitating a larger proportion of CD2. Using co‐capping assays and immunoprecipitations from cell lysates, we show that an association between CD2 and CD5 can be found in rat thymocytes, T lymphocytes and in a thymoma cell line. Possibly, this interaction is a direct one, since CD2 and CD5 transiently expressed in Cos7 cells co‐precipitate each other. Furthermore, using CD2 chimeric proteins containing different domains of CD2, expressed in Cos7 cells as well as in stably transfected Jurkat cells, we show that the interaction between CD2 and CD5 is held at both the intra‐ and extracellular levels, but does not involve the transmembrane domain. The fact that both the extracellular and the cytoplasmic domains of CD2 interact with CD5 suggests a specific and tight association between the two molecules, possibly relevant for the fine‐tuning of signal transduction in T lymphocytes.

A new transmembrane 4 superfamily molecule in the nematode, Caenorhabditis elegans.

Transmembrane 4 superfamily (TM4SF) molecules are predominantly mammalian cell surface glycoproteins that are thought to transduce signals mediating cell development, activation, and motility. Analysis of the Genpept sequence database reveals YKK8, a novel member of the TM4SF in the nematode,Caenorhabditis elegans. YKK8 is a putative 27.4-kDa protein encoded by a gene on chromosome III of theC. elegans genome (Wilson et al. [1994]Nature 368:32–38). The assignment of YKK8 to the TM4SF is justified by three criteria: statistical comparison of protein sequences, conserved TM4SF protein sequence motifs, and conserved TM4SF intron/exon boundaries in the genomic sequence. The discovery of a TM4SF molecule in the nematode extends this superfamily to a more primitive branch of the phylogenetic tree and suggests a fundamental role for TM4SF molecules in biology.

Characterisation of mouse CD37: cDNA and genomic cloning.

The leukocyte surface antigen CD37 is a member of the transmembrane 4 superfamily (TM4SF) of glycoproteins which are predicted to span the lipid bilayer four times. The protein sequence and gene structure of mouse CD37 (Cd37) have been deduced through the isolation of cDNA and genomic clones. The Cd37 gene produces a major mRNA transcript of 1.2 kb that is restricted to cells of lymphoid and myeloid origin. Mouse CD37 is a glycoprotein of 281 amino acids in length, encoded by eight exons that span approximately 5.2 kb. CD37 is highly conserved between species, the mouse sequence sharing amino acid identities of 98% and 79% with rat and human, respectively. Cd37 shows a striking similarity in genomic organisation to other members of the TM4SF, which is consistent with the theory that this superfamily has evolved by gene duplication and divergence from a common ancestral gene.

The molecular characterisation of a novel tetraspanin protein, TM4-B

TM4-B is a novel member of the Tetraspanin superfamily and displays characteristics typical of the superfamily. It bears significant homology to other superfamily members and is most similar to Tspan-1. This molecule is broadly expressed in most human tissues and cell lines including neural and bone marrow derived tissues. TM4-B was mapped to the q34 region on human chromosome 9.