Abstract
Macrosialin is a heavily glycosylated transmembrane protein of 87-115 kDa, highly and specifically expressed by mouse tissue macrophages, and to a lesser extent by dendritic cells. We have isolated cDNA clones encoding macrosialin from a thioglycollate-elicited peritoneal macrophage cDNA library by transient expression in COS cells and panning with the anti-macrosialin monoclonal antibody FA/11. A single 1.3-kilobase macrosialin transcript was detected in both untreated and phorbol 12-myristate 13-acetate-stimulated RAW cells. The cDNA sequence predicts a type I integral membrane protein of 326 residues with a heavily glycosylated extracellular domain of 306 residues containing nine potential N-linked glycosylation sites and numerous potential O-linked glycosylation sites. The extracellular domain consists of two distinct regions, separated by an extended 12 residue proline-rich hinge; a membrane-distal mucin-like domain of 89 residues containing short peptide repeats and consisting of 44% serine and threonine residues; and a membrane proximal domain of 170 residues, which has significant sequence homology to a family of lysosomal associated glycoproteins known as the lamp-1 group. Macrosialin is the murine homologue of the human macrophage glycoprotein CD68 (72% identity, 80% similarity). Both proteins are preferentially expressed by macrophages and share the same bipartite structure having a mucin-like domain and a domain common to the lamp family. Macrosialin and CD68 are the first examples of a lamp family protein with a restricted cell-type-specific expression. They may have evolved from the lamps to carry out specialized functions in dedicated phagocytic cells.
Highlights
Somal associated glycoproteins known as the lamp-1 Macrosialin and CD68 share a number of common biochemgroup.Macrosialin is themurine homologue of the ical properties: macrophage-restricted expression, similar mohuman macrophage glycoprotein CD68(72%identity, lecular mass (87-115 kDa), similar glycosylation patterns, 80%similarity)
We have recently cloned a cDNA encoding CD68’ by transient expression in COS cells and found that it is a member of a family of lysosomal/endosomalssociated glycoproteins known as the lamp/lgp family
To better understand the structure and functioonf macrosialin, and more clearly establish the relationship of human CD68 to murine macrosialin, we report here thecloning of a cDNA clone encoding macro
Summary
CD68 (72% identity). CD68/macrosialin are lineagerestricted, lysosomal-associated glycoproteins that could play roles in the specialized phagocytic activities of tissue macrophages, both in intracellularlysosomal metabolism and extracellular cell-cell and cell-pathogen interactions. 92 "C for 1min, 50 "Cfor 1min, 72 "C for 1min. PCR productswere treated with Klenow and polynucleotide kinase and blunt-end-cloned into phosphatase-treated EcoRV-cut pBluescript. Individual recombinant colonies were sequencedwithuniversaMl 13sequencing primers. DNA Sequencing-Double-stranded sequencing was conducted on the cDNA insert in pMS.l by dideoxy chain termination (Sanger et MATERIALSANDMETHODS al., 1977) using sequence-specific oligonucleotides. Cell Lines and Culture Conditions-Thioglycollate-elicited perito- the cDNA insert were sequenced entirely
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