Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus.
Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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H V
HV
Schering-Plough Research, Bloomfield, New Jersey 07003.
Seelig
G F
GF
Syto
R
R
Ramanathan
L
L
Windsor
W T
WT
Borkowski
D
D
Trotta
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007106:Immune Sera / Q000737:chemistry; D015847:Interleukin-4 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate; D013211:Staphylococcus aureus
D007106:Immune Sera; D010446:Peptide Fragments; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D011994:Recombinant Proteins; D015847:Interleukin-4; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase