Inhibition of degranulation of polymorphonuclear leukocytes by angiogenin and its tryptic fragment.
A degranulation inhibiting protein was purified to apparent homogeneity from plasma ultrafiltrates of patients with uremia using gel-permeation chromatography, ion-exchange chromatography, affinity chromatography on blue Sepharose, and ion-exchange chromatography on a Mono S HR 5/5 fast protein liquid chromatography column. The identity of the isolated degranulation inhibiting protein with angiogenin was demonstrated by amino acid sequence determination, immunoblotting, and identical inhibitory activity effects on leukocyte degranulation. At concentrations in the nanomolar range, the protein inhibited spontaneous degranulation of polymorphonuclear leukocytes (PMNL) to 40%. The protein discharge of cells, which were preincubated with nanomolar concentrations of angiogenin and then stimulated with the chemotactic peptide formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-leucine (FNLPNTL), was inhibited by 70%. Cellular functions such as chemotaxis, phagocytosis, and the oxidative respiratory burst were not obviously affected by angiogenin. A polyclonal antibody to human recombinant angiogenin abolished the inhibitory effect of the isolated protein upon PMNL. The same but reduced effect was induced by the disulfide C39-C92 containing tryptic angiogenin fragment L-H-G-G-S-P-W-P-P-C92-Q-Y-R-G-L-T-S-P-C39-K, indicating a new, so far unknown biologically active site of angiogenin which is different from the sites responsible for angiogenic or ribonucleolytic activity. Two synthetic peptides containing residues 83-95, one with S92 instead of C92, revealed the same inhibitory effect on the protein degranulation of PMNL as the entire tryptic fragment.
Tschesche
H
H
Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany.
Kopp
C
C
Hörl
W H
WH
Hempelmann
U
U
D000595:Amino Acid Sequence; D003488:Cyanogen Bromide; D003594:Cytoplasmic Granules / Q000502:physiology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007676:Kidney Failure, Chronic / Q000097:blood; D007700:Kinetics; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D012016:Reference Values; D012259:Ribonuclease, Pancreatic; D014357:Trypsin
D010446:Peptide Fragments; D011506:Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D003488:Cyanogen Bromide