Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein.
The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP-specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33-kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased.
Popken-Harris
P
P
Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
McGrogan
M
M
Loegering
D A
DA
Checkel
J L
JL
Kubo
H
H
Thomas
L L
LL
Moy
J N
JN
Sottrup-Jensen
L
L
Snable
J L
JL
Kikuchi
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001798:Blood Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D047091:Eosinophil Granule Proteins; D047093:Eosinophil Major Basic Protein; D005822:Genetic Vectors; D006031:Glycosylation; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases; D013481:Superoxides / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002241:Carbohydrates; D047091:Eosinophil Granule Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D013481:Superoxides; C074129:PRG2 protein, human; D013762:Tetrahydrofolate Dehydrogenase; D012260:Ribonucleases; D047093:Eosinophil Major Basic Protein