Biochemical properties, activities, and presence in biologic fluids of eosinophil granule major basic protein.
The existence of a proform of MBP is predicted from the sequence of MBP cDNA clones. ProMBP has been purified from the supernatants of CHO cells transfected with cDNA encoding prepro MBP. Purification involved heparin-Sepharose affinity purification followed by two sequential size fractionation steps over Sephadex G-100 and yielded proMBP with a molecular mass of 33 kd. Recombinant proMBP from the heparin-Sepharose column was subjected to isoelectric focusing followed by SDS-PAGE and Western blot analysis. The results indicated that most of the 33 kd form of proMBP focused predominantly between pI 4.2 and 5.1, with a major peak at a pI of approximately 4.9. Analyses of the carbohydrates associated with the purified 33 kd form of recombinant proMBP indicated the addition of 4856 to 5150 Da by carbohydrates characteristic of the complex type. Consistent with the hypothesis that the function of the propiece is to neutralize MBP toxicity during granule processing, proMBP lacked MBP cytostimulatory properties and actually blocked the effect of MBP in two different systems, basophil histamine release and neutrophil activation. In addition, as a measure of toxicity, proMBP did not inhibit protein synthesis, whereas MBP markedly reduced protein synthesis. The mechanisms by which MBP exerts its actions both as a cytostimulant and as a toxin are not known; however, it is known that cationic MBP readily reacts with acidic lipids. Using artificial liposomes as targets, MBP caused a disordering of the lipid bilayer membrane, resulting in fusion and lysis. Therefore, MBP may act both as a cytostimulant and as a toxin because of its marked cationicity and its ability to disorder lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Popken-Harris
P
P
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
Thomas
L
L
Oxvig
C
C
Sottrup-Jensen
L
L
Kubo
H
H
Klein
J S
JS
Gleich
G J
GJ
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis / Q000502:physiology*; D047091:Eosinophil Granule Proteins; D047093:Eosinophil Major Basic Protein; D004804:Eosinophils / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000032:analysis / Q000502:physiology; D011994:Recombinant Proteins; D012260:Ribonucleases
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; C074129:PRG2 protein, human; D012260:Ribonucleases; D047093:Eosinophil Major Basic Protein