Chemical modification of interleukin-1 beta: biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein.
We have modified recombinant human Interleukin-1 beta using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide at pH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI = 6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1 beta. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 beta and may be important for the binding of IL-1 to its receptor.
Yem
A W
AW
Upjohn Company, Kalamazoo, Michigan 49001.
Guido
D M
DM
Mathews
W R
WR
Staite
N D
ND
Richard
K A
KA
Prairie
M D
MD
Krueger
W C
WC
Epps
D E
DE
Deibel
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D007375:Interleukin-1 / Q000737:chemistry* / Q000494:pharmacology; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013601:T-Lymphocytes / Q000166:cytology
D003432:Cross-Linking Reagents; D007375:Interleukin-1; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D005022:Ethyldimethylaminopropyl Carbodiimide