Abstract

C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.

Highlights

  • C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins

  • Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown

  • We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy

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Summary

ARTICLE cro

The cartilage-specific lectin C-type lectin domain family 3 member A (CLEC3A) enhances tissue plasminogen activator–mediated plasminogen activation. We have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation. Whereas tetranectin binds to the plasminogen kringle 4 [3] and is thought to enhance tissue plasminogen activator (tPA)-mediated plasminogen activation [4], SCGF (CLEC11A) has been described to be a growth factor stimulating hematopoietic progenitor cells [5]. We recombinantly expressed murine and human fulllength CLEC3A as well as truncated forms comprising the potential ␣-helical oligomerization domain (OD) and the CRD (CLEC3A-Ex23) or only the CRD (CLEC3A-Ex3) in HEK-293 EBNA cells and generated specific antisera. We studied the interaction of human CLEC3A and plasminogen by enzyme-linked immunosorbent binding assay (ELISA-style binding assay) and by surface plasmon resonance and analyzed the tPA-mediated activation of plasminogen in the presence of human CLEC3A by plasminogen conversion assay

Results
Antibody generation
Discussion
Experimental procedures
Synthetic peptides
Glycan analysis
Affinity purification
Circular dichroism spectroscopy
Tissue extraction
Immunofluorescence microscopy
Surface plasmon resonance
Plasminogen activation assay
Full Text
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