Molecular cloning of a human macrophage lectin specific for galactose.

Abstract

The murine Mac-2 protein is a galactose-and IgE-binding lectin secreted by inflammatory macrophages. We describe here the cloning and characterization of a cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive prolineglycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes.These findings are discussed in the context of the potential functions of hMac-2.Macrophages activated during the course of acute or chronic inflammation release a number of soluble proteins which mediate or regulate the effects of the inflammatory response. Understanding the function and mode of regulation of these monokines is critical to devising strategies for therapeutic intervention in inflammatory disorders. The murine Mac-2 antigen was originally described by Ho and Springer (1) and was shown to be expressed at a high level on the surface of inflammatory macrophages. We recently cloned cDNAs encoding Mac-2 and showed that the protein is secreted and has the characteristics of a galactose-specific lectin (2). A search ofthe computer data bases revealed that it had been identified independently by two other groups on the basis of its carbohydrate-binding property. Wang and colleagues (3,4) IgE (6). We have shown that Mac-2 also has the ability to bind murine IgE (2).Recently, yet another function has been revealed by the work of Woo et al. (7), who showed that Mac-2 is the major nonintegrin laminin-binding protein synthesized by murine inflammatory macrophages, indicating a potential role in macrophage-extracellular matrix interactions.Given the number of functions and subcellular locations proposed for Mac-2, further studies are clearly warranted. The high expression of this protein in inflammatory macrophages suggests that it has an important function in inflammation. In view of the potential involvement of this protein in processes relevant to human disease, we were interested in determining whether a human homolog of Mac-2 existed. In the present report we describe the cloning of the human homolog of Mac-2 (which we have designated hMac-2, for human Mac-2 antigen)* and show that the primary structure of the two proteins is highly conserved, especially in the lectin domain. The hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 (1) and behaves like a galactose-specific lectin in its binding to the desialylated glycoprotein asialofetuin. It also binds to purified laminin, thus confirming and extending the results of Woo et al. (7). MATERIALS AND METH...

Galectin-3 Marks Activated Macrophages in Failure-Prone Hypertrophied Hearts and Contributes to Cardiac Dysfunction

“…17 It belongs to a lectin family sharing the jelly-roll-like folding pattern and calcium-independent specificity to ␤-galactosides as well as proteins. 18,19 In addition to its antiapoptotic and growth-promoting actions, galectin-3 plays a critical role in phagocytosis by macrophages when cross-linked by the Fc␥ receptor.…”

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

“…The M3/38 monoclonal antibody 19 used in the immunostaining procedure was prepared from the supernatant of the M3/38.1.2.8.HL.2 rat hybridoma TIB 166 obtained from the American Type Culture Collection (Bethesda, MD). Immunohistochemical staining was performed using a nonbiotin amplification system (NBA; Zymed Immunochemicals, South San Francisco, CA).…”

The second intron of the human galectin‐3 gene has a strong promoter activity down‐regulated by p53

“…1 pug of DNA of the ligation ieaction was used as the template for PCR which was carried out in a DNA Thermocvcler (Perkin-ElmerCetus, Norwalk, CT) in a reaction mixture containing '1. 25 [4,6,8] (Fig. 1).…”

“…Thirty amplification cycles were performed (1 min denaturation at 94"C, 2 min annealing at 55°C and 3 min extension at 72°C). 2 ~1 of the reaction were used as the template for a second round of PCR using two nested primers M13F, 5'-GTAAAACGACGGCCAGT-3', located on pUC18 (nt 379-395) and GAL67, 5'-ATCCTTGAGGGTTTGGGTTT-3', located 45-64 bp downstream of the ATG translation initiation codon on the galectin-3 cDNA [4,6,8]. 1 ~1 of this second PCR reaction was used for ligation with a linearized pCRlOO0 plasmid vector and transformation of Escherichia coli as recommended by the manufacturer (TA cloning system; Invitrogen Corp., San Diego, CA).…”

“…On the basis of cDNA sequencing, all galectins-3 are closely homologous, namely CBP35 from 3T3 fibroblasts [2], L-31 and L-34 from human and murine tumour cells [3,4], Mac-2 from human and rat macrophages [5,6], Ig-E binding protein or E BP from basophilic leukaemia cells [7,8], and L-29 in human and rat lung [9,10]. The protein can be found intracellularly in the cytosol and in the nucleus and extracellularly in the medium and at the cell surface [3,1 l-141, suggesting its involvement in various biological processes.…”