Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1.
BACKGROUND
BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils.


OBJECTIVE
We sought to further characterize the basophilspecific antigen identified by BB1.


METHODS
Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing.


RESULTS
In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6.


CONCLUSIONS
With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.
McEuen
A R
AR
Immunopharmacology Group, Southampton General Hospital, UK.
Calafat
J
J
Compton
S J
SJ
Easom
N J
NJ
Buckley
M G
MG
Knol
E F
EF
Walls
A F
AF
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000032:analysis; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001491:Basophils / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D019921:Biotinylation; D001797:Blood Protein Electrophoresis; D002460:Cell Line; D002850:Chromatography, Gel; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006967:Hypersensitivity / Q000378:metabolism; D007150:Immunohistochemistry; D007249:Inflammation / Q000378:metabolism; D018836:Inflammation Mediators / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008854:Microscopy, Electron; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011402:Pronase / Q000494:pharmacology; D060149:Tetraspanin 30
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000954:Antigens, Surface; C554586:CD63 protein, human; D018836:Inflammation Mediators; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D011402:Pronase