Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1. Two L1 isoforms in the cytoplasmic region are produced by differential splicing

We have isolated and sequenced a full-length cDNA encoding the rat neural cell adhesion molecule L1. The deduced amino acid sequence as a whole shows high homology to mouse L1 sequence. In addition to this complete form of L1, we found an isoform, L1cs, which lacks four amino acid residues (RSLE) in the cytoplasmic domain and probably is derived from the same single L1 gene by tissue-specific alternative splicing. While L1 mRNA was predominantly expressed in the brain, L1cs mRNA was found exclusively in peripheral nervous tissue. Differential splicing in the highly conserved cytoplasmic domain may play an important role in modulating the function of L1 in different cells.     

Identification of the Tslc1 gene,a mouse orthologue of the human tumor suppressor TSLC1 gene

We have recently identified the TSLC1 gene as a novel tumor suppressor in human non-small lung cancer on chromosome 11q23.2. TSLC1 encodes a membrane glycoprotein showing significant homology with immunoglobulin superfamily molecules. Here, we report the isolation of a mouse orthologous gene, Tslc1. The Tslc1 cDNA contains a single open reading frame of 1335 bp encoding a putative protein of 445 amino acids, and its expression was detected in all tissues examined. The Tslc1 gene is mapped on mouse chromosome 9, a synteny of human chromosome 11q, and is composed of ten exons, the exon-intron junctions being highly conserved between human and mouse. The predicted amino acids of mouse Tslc1 display 98% identity with that of human TSLC1. Furthermore, data base analysis indicates that the amino acid sequences corresponding to the cytoplasmic domain of Tslc1 are identical in five mammals and highly conserved in vertebrates, suggesting an important role of Tslc1 in normal cell-cell interaction.     

Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region

We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequences identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40 Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.     

Characterization of a new ATP-binding cassette transporter in Trypanosoma cruzi associated to a L1Tc retrotransposon

We have characterized the tcpgp1-like gene of Trypanosoma cruzi, a new ATP-binding cassette (ABC) transporter. tcpgp1 codes for a 1035 amino acid protein with a considerable homology to LtpgpA of Leishmania. Tcpgp1 lacks the conserved sequences corresponding to the second nucleotide-binding domain of other ABC transporters due to the insertion of the L1Tc non-LTR retrotransposon.     

Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.

Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.     

Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants.

The sialic acid binding immunglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within the Siglec family, there exists a subgroup, which bears a high degree of homology with the molecule CD33 (Siglec-3), and has thus been designated the CD33-like subgroup of Siglecs. Members of this subgroup have been localized to chromosome 19q13.4. Through the positional candidate approach, we identified a novel potential member of this subgroup of Siglecs. We have characterized the complete genomic structure of this gene, determined its chromosomal localization, its homology to other members of the Siglec family, and its tissue expression profile. This new Siglec-like gene is comprised of 11 exons, with 10 intervening introns, and is localized 278 kb telomeric to Siglec-9 and 35 kb centromeric to Siglec-8 and on chromosome 19q13.4. The coding region consists of 2094 base pairs, and encodes for a putative 76.6 kDa protein. All Siglec-conserved structural features, including V-set domain, three C-set domains, transmembrane domain, ITIM and SLAM motifs, were found in this Siglec-like gene. Also, it has the conserved amino acids essential for sialic acid binding. The Siglec-like gene has 40-66% homology with members of the CD33-like subgroup, including Siglecs 5-9. Through RT-PCR we have examined the expression profile of this new gene in a panel of human tissues and found it to be primarily expressed in the bone marrow, spleen, brain, small intestine, colon, and spinal cord. We were also able to identify three different splice variants of the new gene. This gene may represent the latest novel member of the CD33-like subgroup of Siglecs, and, given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec-7.     

Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model

VopF, the type III effector molecule, has been implicated in the pathogenesis of non-O1, non-O139 strains of Vibrio cholerae. It is a protein of 530 amino acids, comprises of one formin homology 1-like (FH1-like) domain and three WASP homology 2 (WH2) domains. Previous works have demonstrated that WH2 domains are crucial for VopF function as a modulator of cellular actin homeostasis. Furthermore, domain deletion analysis also suggests that VopF variant constituted with only WH2 domain 3 is more efficient in restricting the growth of budding yeast than its congeners containing either only domain 1 or domain 2. Interestingly, a good degree of sequence diversity is present within each WH2 domain of VopF. In order to ascertain the importance of different amino acids in each WH2 domain, a systemic alanine scanning mutagenesis was employed. Using a yeast model system, the alanine derivatives of each amino acid of WH2 domain 1 and 3 of VopF were examined for growth restricting activity. Taken together, our mutagenesis results reveal the identification of critical residues of WH2 domain 1 and 3 of VopF.     

The LRR and Ig domain-containing membrane protein SST273 is expressed on motoneurons

We performed signal sequence trap cDNA cloning using a cDNA library of enriched chicken embryonic spinal motoneurons and identified a novel transmembrane molecule, SST273 (Signal Sequence Trap clone 273). SST273 has five leucine-rich repeats (LRRs) and one immunoglobulin (Ig) domain in its extracellular domain. The human (KIAA1465) and mouse (BC059068) homologues of SST273 were already cloned, but had not yet been analyzed. The amino acid homologies between chick SST273 and human or mouse homologue are 52.1 and 51.5%, respectively. SST273 mRNA and its protein product were uniquely expressed in the embryonic spinal and cranial motoneurons at early developmental stages.     

Evolution and mutagenesis of the mammalian excision repair gene ERCC-1.

The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E. coli repair proteins uvrA and uvrC. To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue. Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent. Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible. The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain. The ERCC-1 promoter harbors a region which is highly conserved in mouse and man. Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families

Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence.     

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 characterization of a new member of the immunoglobulin superfamily that potentially functions in T-cell activation and development

 Differential hybridization cloning has been used to isolate a novel chicken thymic activation and developmental sequence (cTADS). The nucleotide sequence of the cTADS cDNA predicts an open reading frame of 439 amino acids. The inferred cTADS protein possesses a hydrophobic membrane-spanning domain and putative intracellular kinase activation domains. Its extracellular domain shares similarities with the immunoglobulin protein superfamily, featuring two conserved immunoglobulin folds that resemble C1 and C2 constant regions. The cTADS sequence shows similarity to a subfamily of proteins involved in cellular adhesion: chicken neural cell adhesion molecule and human opioid-binding adhesion molecule, and to proteins that have a biological role in intracellular signaling: mouse platelet-derived growth factor receptor and human fibroblast growth factor receptor. cTADS is differentially expressed in chicken thymic cells during embryonic development and during activation through the T-cell receptor. Sequence similarities and expression patterns suggest that cTADS could be involved in cell recognition and adhesion, and/or peptide ligand binding. Received: 1 May 1999 / Revised: 1 October 1999     

L1-mediated axon outgrowth occurs via a homophilic binding mechanism

The molecular mechanism by which the L1 cell adhesion molecule mediates neurite outgrowth has been examined. Purified L1 from mouse and L1 from chick brain were attached to nitrocellulose dishes. Both chick and mouse neurons were able to adhere to purified mouse L1 and chick L1. Both molecules promoted neurite extension from chick and mouse neurons. Addition of Fabs specific for chick L1 to the cultures inhibited chick neurite outgrowth on both mouse L1 and chick L1. These findings suggest that L1-like molecules support neurite outgrowth via a "homophilic" binding mechanism.