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Journal of Biochemistry Advance Access originally published online on December 13, 2006
Journal of Biochemistry 2007 141(1):127-136; doi:10.1093/jb/mvm017
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© 2006 The Japanese Biochemical Society.

Properties of Blocking and Non-blocking Monoclonal Antibodies Specific for Human Macrophage Galactose-type C-type Lectin (MGL/ClecSF10A/CD301)

Yoshihiko Sano, Katsuaki Usami, Ryota Izawa, Kaori Denda-Nagai, Nobuaki Higashi, Toshifumi Kimura, Noriko Suzuki and Tatsuro Irimura*

Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0003, Japan

*To whom correspondence should be addressed. Tel: +81-3-5841-4870, Fax: +81-3-5841-4879, E-mail: irimura{at}mol.f.u-tokyo.ac.jp

Received August 24, 2006; Accepted November 7, 2006


  Abstract

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.

Key Words: CD301/MGL, dendritic cell, lectin, monoclonal antibody, viral infection

Abbreviations: BSM, bovine submaxillary mucin; CRD, carbohydrate recognition domain; C-type lectin, calcium-type lectin; DPBS, Dulbecco's PBS; ECL, enhanced chemiluminescence; GalNAc, N-acetylgalactosamine; HRP, horseradish peroxidase; mAb, monoclonal antibody; MGL, macrophage galactose-type C-type lectin; MGL-COS-1 cells, COS-1 cells transfected with MGL cDNA; Mock-COS-1, mock transfected COS-1 cells; MOPS, 3-(4-morpholino) propane sulfonic acid; TBST, Tris-buffered saline containing 0.1% Tween-20; PVDF, polyvinylidene difluoride


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