Identification of paramyosin as a binding protein for calgranulin C in experimental helminthic keratitis.
OBJECTIVE
Calgranulin C (CaGC) is a protein released by activated neutrophils and involved in host defense against filarial infections. This study involved the identification of binding protein(s) of the helminth Brugia malayi to CaGC and the ability of binding complexes to induce keratitis.


METHODS
Parasitic extracts prepared from B. malayi microfilariae and adult worms were incubated with recombinant CaGC protein. Parasite binding protein-CaGC complex was isolated by affinity chromatography. A B. malayi microfilariae cDNA library was immunoscreened with antisera from rats immunized with the isolated parasitic CaGC-binding protein. All positive clones contained paramyosin sequences. Paramyosin was thus considered the major CaGC-binding protein in the parasite. To delineate the binding of CaGC to native and recombinant paramyosin, 125I-CaGC was used as a binding tracer in SDS-PAGE analysis to identify a CaGC-binding complex. To determine whether the complex of CaGC and its binding protein could induce keratitis mimicking the onchocercal human corneal disease, BALB/c mice preimmunized with the binding complex were challenged with intracorneal binding complex or live Brugia microfilariae. In addition, splenocytes harvested from the same animals were assessed for their ability to elicit cellular immune responses to the binding complex by [3H]thymidine assay.


RESULTS
In vitro binding of CaGC to paramyosin was confirmed using recombinant paramyosin and 125I-CaGC. Test animals showed development of severe keratitis that mimicked, clinically and histopathologically, the human onchocercal corneal disease, demonstrating the antigenic specificity of the paramyosin-CaGG-binding complex.


CONCLUSIONS
Paramyosin is identified as a CaGC-binding protein in B. malayi.
Akpek
Esen K
EK
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.
Liu
Sammy H
SH
Thompson
Robert
R
Gottsch
John D
JD
D000818:Animals; D017178:Brugia malayi / Q000502:physiology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015822:Eye Infections, Parasitic / Q000378:metabolism* / Q000469:parasitology / Q000473:pathology; D005260:Female; D005368:Filariasis / Q000378:metabolism* / Q000469:parasitology / Q000473:pathology; D007111:Immunity, Cellular; D007114:Immunization; D007634:Keratitis / Q000378:metabolism* / Q000469:parasitology / Q000473:pathology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D015800:Protozoan Proteins / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins; D009418:S100 Proteins / Q000276:immunology / Q000378:metabolism*; D000067616:S100A12 Protein; D014335:Tropomyosin / Q000276:immunology / Q000378:metabolism*
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009418:S100 Proteins; D000067616:S100A12 Protein; C090441:S100A12 protein, human; D014335:Tropomyosin