Human Siglec-5: tissue distribution, novel isoforms and domain specificities for sialic acid-dependent ligand interactions.
Human Siglec-5 is a sialic acid binding immunoglobulin (Ig)-like lectin (Siglec), comprising one N-terminal IgV-SET domain followed by three IgC2-SET domains, and a cytoplasmic domain with ITIM and SAP motifs which regulate cell signalling. We report the differential distribution of hSiglec-5 on neutrophil and macrophage subsets in tissues using monoclonal antibodies, 1A5 and 2H8, which require the first IgC2-SET domain for binding. Interestingly, hSiglec-5 was especially prominent on macrophages in reactive lymph nodes. We have identified four isoforms of hSiglec-5 possessing three (hSiglec-5-3L and -3C) or four (hSiglec-5-4L and -4S) extracellular domains linked to long (hSiglec-5-3L and -4L) or short (hSiglec-5-4S) cytoplasmic tails or existing as a soluble isoform (hSiglec-5-3C). hSiglec-5-4L has the broadest tissue distribution, being detected in adult spleen, thymus, lymph node, peripheral blood leucocytes and bone marrow, and in fetal lung and liver. A soluble Fc chimaeric protein containing the hSiglec-5-4L extracellular domain binds in a sialic acid-dependent manner to glycophorin A on human erythrocytes and to alpha2-3- and alpha2-6-sialyllactose moieties. Domain deletion mutants of hSiglec-5(D1-4)-Fc reveal that the first three IgC2-SET domains are required for optimal binding, with adhesion being abolished if the first IgC2-SET domain is deleted. This indicates that each hSiglec-5 isoform will interact with sialic acid ligands and provides the first step towards defining structure-function relationships of hSiglec-5 isoforms.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002678:Chimera; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D004912:Erythrocytes / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000939:Epitopes; D037102:Lectins; D020033:Protein Isoforms; C114531:SIGLEC5 protein, human; D019158:N-Acetylneuraminic Acid