Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein.
Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.
Thomas
L L
LL
Department of Immunology/Microbiology, 1653 West Congress Parkway, Rush Medical College, Chicago, IL 60612, USA. lthomas2@rush.edu
Kubo
H
H
Loegering
D J
DJ
Spillard
K
K
Weaver
A J
AJ
McCormick
D J
DJ
Weiler
C
C
Gleich
G J
GJ
D000595:Amino Acid Sequence; D001491:Basophils / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000502:physiology*; D003602:Cytotoxicity, Immunologic; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology*; D006632:Histamine / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D017997:Leukotriene C4 / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology / Q000502:physiology*; D010449:Peptide Mapping / Q000379:methods; D012260:Ribonucleases; D020539:Sequence Analysis, Protein / Q000379:methods; D014407:Tumor Cells, Cultured
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D010446:Peptide Fragments; D017997:Leukotriene C4; D006632:Histamine; D012260:Ribonucleases