Identification of a reactive lysyl residue (Lys103) of recombinant human interleukin-1 beta. Mechanism of its reactivity and implication of its functional role in receptor binding.
Recombinant human interleukin-1 beta (h-IL-1 beta) was chemically modified with 4-(N,N-dimethylamino)-4'-isothiocyanatoazobenzene-2'-sulfonic acid (S-DABITC), a water-soluble color reagent specific for lysine labeling. Modified h-IL-1 beta was digested by lysyl endopeptidase. Peptides containing labeled lysines were detected at the visible wavelength (436 nm) and isolated by HPLC. The modification sites were eventually determined by sequence analysis. The results revealed that Lys103, Lys92, Lys93, and Lys94 of h-IL-1 beta reacted selectively with S-DABITC. A 1-h incubation with 1 mM S-DABITC at room temperature resulted in a quantitative modification of Lys103, 22% of Lys92, 27% of Lys93, and 18% of Lys94, respectively. This modification was accompanied by a 20-fold decrease of the protein's ability to bind to the receptor. Furthermore, a mutant of h-IL-1 beta (M9, Glu105 substituted by Lys) exhibits markedly impaired receptor binding, and the S-DABITC reactivity of its Lys103 was found to be reduced by 90%. These findings suggest that Lys103 of h-IL-1 beta might play an important role in the h-IL-1 beta/receptor interaction.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Ngai
P K
PK
Priestle
J P
JP
Joss
U
U
Vosbeck
K D
KD
van Oostrum
J
J
D000595:Amino Acid Sequence; D002448:Cell Adhesion; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D007202:Indicators and Reagents; D007375:Interleukin-1 / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D017879:Isothiocyanates; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011971:Receptors, Immunologic / Q000378:metabolism*; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship; D013861:Thiocyanates; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D007202:Indicators and Reagents; D007375:Interleukin-1; D017879:Isothiocyanates; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D013861:Thiocyanates; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D008239:Lysine