A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge.
A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K(i)) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein. On SDS-electrophoresis, gBrEI appeared as a single 20-kDa band, also after reduction. Schiff reagent staining indicated a carbohydrate portion in the protein, which was confirmed by mass spectrometry. The glycosylated site was Asn 38, and a carbohydrate portion of 1.17 kDa was identified. gBrEI was found to contain 144 amino acid residues, and a FASTA database analysis showed that it belongs to the plant Kunitz-type inhibitor family. Val66 was identified as reactive site P1 residue by comparison of conserved positions in the sequences. Since gBrEI harbors a single disulfide bridge, it may be considered a new type of Kunitz inhibitor, intermediate between the classical Kunitz inhibitors, which contain two disulfide bridges, and those from B. bauhinioides, which do not have such bridges.
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, Brazil.
Joana Tomomi
JT
Sumikawa
Adriana Miti
AM
Nakahata
Hans
H
Fritz
Reinhardt
R
Mentele
Misako Uemura
MU
Sampaio
Maria Luiza
ML
Oliva
D000595:Amino Acid Sequence; D000818:Animals; D036481:Bauhinia; D002417:Cattle; D004220:Disulfides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000187:drug effects; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D012639:Seeds; D013552:Swine
D004220:Disulfides; D004791:Enzyme Inhibitors; D010936:Plant Extracts; D010196:Pancreatic Elastase