Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine, thiol and aspartyl proteases.
We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Galbeta)Ser or (Glcalpha,beta)Tyr at P2 or P3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8-His-Leu-Val-Ile-His-Asn14 of human angiotensinogen, in which [GlcNAcbeta]Asn was introduced before Phe8 and/or after His13 and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal3)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glcalpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P'4 glycosylated peptide [Abz-F-H-L-V-I-H-(GIcNAcbeta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K(m) and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P'4 and P4 glycosylated substrates.
Juliano
M A
MA
Department of Biophysics, Escola Paulista de Medicina, São Paulo, Brazil.
Filira
F
F
Gobbo
M
M
Rocchi
R
R
Del Nery
E
E
Juliano
L
L
D000808:Angiotensinogen / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D012083:Renin / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D020840:Tissue Kallikreins; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D000808:Angiotensinogen; D007610:Kallikreins; D012697:Serine Endopeptidases; D020840:Tissue Kallikreins; D014357:Trypsin; D003546:Cysteine Endopeptidases; D010206:Papain; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; D012083:Renin; D002402:Cathepsin D