CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells

To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 3365-3369) to allow cloning of intracellular antigens. A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes. Individual cells expressing intracellular CD63 were identified by autoradiography. cDNA was extracted from positive spots and reintroduced into Escherichia coli. After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis. The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites. The CD63 gene was expressed in a wide variety of cells. Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955-2962). By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein. CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.     

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes

Partial cDNA sequences of both CD8beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8beta and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8beta is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8betas, carp CD8beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp.     

Identification and characterisation of a homolog of an activation gene for the recombination activating gene 1 (RAG 1) in amphioxus

Expression of recombination activating genes (RAG) involved in the V (D) J recombination is regulated by the RAG1 gene activator (RGA) in mammals. The sequence of a cDNA clone from an amphioxus cDNA library was found to be homologous to that of RGA from mouse stromal cells with 45% identity. The full-length cDNA sequence comprises 1119 bp and encodes a putative protein of 210 amino acid residues. Characterisation of the amino acid sequence revealed that two MtN3 domains and seven transmembrane spans are present in this protein, indicating a potential role as a plasma membrane protein. This gene is expressed in many tissues and at differential developmental stages. A high expression level of RGA is detected in gonad tissues, and gastrula embryo and adult stages. The presence of the RGA gene in amphioxus suggests that the signal pathway required for the expression of RAG could exist in this primitive protochordate. It also implies that in the related molecules, primitive adaptive immunity may have existed in cephalochordate although the complete machinery of VDJ rearrangement may not be formed.     

Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver

Glutathione-S-transferases have been identified in all the living species examined so far, yet little is known to date about them in amphioxus, a model organism for insights into the origin and evolution of vertebrates. We have isolated a cDNA encoding an amphioxus (Branchiostoma belcheri) glutathione-S-transferase with a predicted molecular mass of approximately 26 kDa, from the gut cDNA library. The glutathione-S-transferase had 43.7-51.8% identity to most glutathione-S-transferases identified from aquatic organisms including fish and green alga, but it was much less identical (<27%) to other cytosolic glutathione-S-transferase classes. The phylogenetic analysis revealed that the glutathione-S-transferase was grouped together with most piscine and algal glutathione-S-transferases, separating from other cytosolic glutathione-S-transferase classes. Moreover, the glutathione-S-transferase had an exon-intron organization typical of zebrafish putative GST, red sea bream GSTR1 and plaice GSTA1 genes. The recombinant glutathione-S-transferase has been successfully expressed and purified, which showed a relatively high catalytic activity (3.37+/-0.1 unit/mg) toward 1-chloro-2, 4-dinitrobenzene and a moderate activity toward ethacrynic acid (0.41+/-0.01 unit/mg), although it had no detectable activity toward 1, 2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 4-nitrobenzyl chloride and cumene hydroperoxide. In addition, we have revealed a tissue-specific expression pattern of the glutathione-S-transferase gene in B. belcheri, with the most abundant expression in the hepatic caecum. All these indicate that the amphioxus glutathione-S-transferase belongs to a novel rho-class of glutathione-S-transferases with a tissue-specific expression pattern. The relation between the glutathione-S-transferase expression in amphioxus hepatic caecum and the origin of vertebrate liver is also discussed.     

Cloning and characterization of NM23-Bbt2 gene from amphioxus Branchiostoma belcheri tsingtauense

A full-length amphioxus (Branchiostoma belcheri tsingtauense) NM23-Bbt2, NM23-H2 homologue, cDNA was isolated from the cDNA library and sequenced. The obtained amphioxus NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species, and all of them contained highly conserved motifs that play important roles in the function of NM23. RT-PCR revealed that NM23-Bbt2 is expressed in the neuronal tissues and is expressed in all stages during the embryogenesis. Nucleoside kinases are thought to have a critical role in regulatory processes such as signal transduction, proliferation, and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in embryogenic development in amphioxus. Phylogenetic analysis showed that the amphioxus group 1 NDPKs (Bf1-4) may be precursors of the human group 1 NDPKs, NM23-Bf5, NM23-Bf6, NM23-Bf7 and NM23-Bf8 may be precursors of NM23-H5, NM23-H6, NM23-H7 and M23-H8, respectively. Our finding of nine NM23 genes in Branchiostoma floride, the precursor of vertebrates, strongly suggests that the ancestral gene corresponding to each of vertebrates NM23 genes generated before the appearance of vertebrates. Comparison of the gene structures of NM23-H2 homologue from invertebrates to vertebrates suggests that the locations of three of the four introns are conserved in amphioxus and vertebrates.     

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.     

Colinear and segmental expression of amphioxus Hox genes.

The cephalochordate amphioxus has a single Hox gene cluster. Here we describe the genomic organization of four adjacent amphioxus genes, AmphiHox-1 to AmphiHox-4, together with analysis of their spatiotemporal expression patterns. We demonstrate that these genes obey temporal colinearity and that three of the genes also obey spatial colinearity in the developing neural tube. AmphiHox-1, AmphiHox-3, and AmphiHox-4 show segmental modulation of their expression levels, a two-segment phasing of spatial colinearity, and, at least for AmphiHox-4, asymmetrical expression. AmphiHox-2 is unlike other amphioxus Hox genes: it does not obey spatial colinearity and it has no positional expression in the neural tube. AmphiHox-2 is expressed in the preoral pit of larvae, from which the homologue of the anterior pituitary develops. We suggest that the ancestral role of chordate Hox genes was primarily in the neural tube and that chordate Hox genes can functionally diverge in a manner analogous to that of Drosophila ftz or zen.     

The 21-kDa Polypeptide (VAP21) in the Rabies Virion Is a CD99-Related Host Cell Protein

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.     

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.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Identification and tissue-specific expression of a promethin-like homolog in amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.     

Cutting edge: primary structure of the light chain of fusion regulatory protein-1/CD98/4F2 predicts a protein with multiple transmembrane domains that is almost identical to the amino acid transporter E16

The CD98 light chain (CD98LC) was copurified from HeLa S3 cells by an affinity chromatography using a mAb specific for the fusion regulatory protein-1 (FRP-1) which is identical to the CD98 heavy chain. On the basis of the N-terminal sequence (63 amino acids) of purified CD98LC polypeptide, we have cloned a PCR fragment (155 bp) from a HeLa S3 cDNA library and finally obtained a full cDNA clone encoding the CD98LC. Fluorescence in situ hybridization analysis using the cDNA assigned the CD98LC gene to the long arm of human chromosome 16 (16q24). The predicted amino acid sequence suggested that CD98LC is a protein with multiple transmembrane domains and is almost identical to the amino acid transporter E16. Resting monocytes and lymphocytes expressed CD98LC as analyzed by a newly isolated anti-CD98LC mAb, which showed cross-reactivity with insect Sf9 cells as well as with various mammalian cell lines.     

大黄鱼CD53基因克隆和分子特征分析

白细胞表面抗原CD53属于四跨膜蛋白超家族,在免疫反应中起着重要作用。在构建大黄鱼（Larim ichthyscrocea）肌肉组织cDNA文库的基础上,克隆了CD53基因。克隆到的CD53基因全长1210bp,其中5-′UTR 113bp,3′-UTR 422bp,CDS 675bp,编码224个氨基酸。生物信息学分析显示大黄鱼CD53存在4次跨膜结构,N端和C端都位于细胞膜内,膜外有两个亲水环,跨膜区域为疏水区域,两个N-糖基化位点都位于靠近C端的亲水环上。大黄鱼CD53氨基酸序列具非常高的保守性,与三刺鱼、斑马鱼、虹鳟等多种鱼类的相似性在70%以上。在检测的10种组织中,CD53只在大黄鱼的脾、骨骼肌、肾、肝、肠组织中表达,其中在肠组织中表达最强。     

Tissue- and stage-specific expression of a fatty acid binding protein-like gene from amphioxus Branchiostoma belcheri

A cDNA clone encoding an amphioxus fatty acid binding protein-like (AmphiFABPL) protein was isolated from a gut cDNA library of Branchiostoma belcheri. It contained a 423 bp open reading frame corresponding to a deduced protein of 140 amino acids with a predicted molecular mass of approximately 15.9 kDa. Phylogenetic analysis showed that AmphiFABPL fell outside the vertebrate clade of fatty acid binding proteins (FABPs), being positioned at the base of the chordate lineage, and was almost equally homologous to various vertebrate FABPs, suggesting that it may be the archetype of vertebrate FABPs. Both northern blotting and in situ hybridization analyses demonstrated that AmphiFABPL was expressed in the hepatic caecum and hind-gut, and although at a much lower level, it was also present in the endostyle, ovary and testis. In addition, whole-mount in situ hybridization revealed that AmphiFABPL was initially expressed in the posterior two thirds of the primitive gut, including the mid-gut where the hepatic caecum will form later, in 2-day larvae. The expression pattern is closely similar to that of the L-FABP and I-FABP genes in vertebrates, supporting the hypothesis that the hepatic caecum in the amphioxus is homologous to the vertebrate liver.