Abstract
The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to alpha2-3 and alpha2-6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galbeta1-4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3-13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway.
Highlights
The Siglecs are a subfamily of I-type lectins that recognize sialic acids
Cloning of a Full-length cDNA Encoding the Putative Siglec9 —During a 5Ј-rapid amplification of cDNA ends experiment that eventually resulted in the cloning of Siglec-7 [20], we noted two independent but highly homologous DNA fragments, one of which turned out to be Siglec-7 and the other an as yet unknown gene product
The most prominent examples are a typically placed arginine (Arg120) and an aromatic amino acid (Trp128), which are known to be involved in sialic acid recognition of Siglec-1/sialoadhesin from x-ray crystallographic analysis of a Siglec-ligand co-crystal [41] and in Siglecs-1, -2, -3, -4, and -7 by mutagenesis experiments [10, 11, 20, 22, 42]
Summary
Materials—Biotinylated polyacrylamide (PAA-Bio) polyvalently substituted with sialylated oligosaccharides were purchased from Glycotech. Homology Search for Siglecs in Fruit Fly and Nematode Genomic Sequences—The sequences of the first 150 amino acids of Siglecs 1–9 (encoding the first Ig V-set domain in each case) were used as templates in homology search of the nr, htgs, and Drosophila genome divisions of GenBankTM data base at the National Center for Biotechnology Information web site using tblastn program [38] These divisions include all cDNA and genomic DNA sequences made public. Putative protein-coding sequences were either found in annotated GenBankTM entries or using Berkeley Fly Data Base of Berkeley Drosophila Genome Project (for fruit fly genomic DNA sequences) These putative protein-coding sequences, as well as the cDNA sequences that showed significant homology (expectation value, Ͻ1) to Siglecs, were translated to amino acid sequences and analyzed for domain structures using the SMART program at European Molecular Biology Laboratories at the Heidelberg web site [39, 40]. Putative orthologs were aligned using ClustalW and analyzed by PAUP 4.0 as described
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
