De novo sequencing and characterization of a novel Bowman-Birk inhibitor from Lathyrus sativus L. seeds by electrospray mass spectrometry.
Bowman-Birk serine protease inhibitors (BBIs) from legume seeds are small proteins showing a two-head structure with distinct reactive site loops, which inhibit two molecules of the same enzyme or two different proteases. Purification and characterization of new BBIs is of broad interest for understanding the basic molecular mechanisms underlying natural defence against the action of proteolytic enzymes. In this study, two novel acidic BBIs (LSI-1a and LSI-2a) were isolated from L. sativus seeds using classical biochemical techniques and characterized for their inhibitory activity. In addition, the N-terminal sequencing of LSI-1a was performed by Edman degradation up to residue 10 and the complete primary structure of the most abundant form (LSI-2a) was determined by using a combination of mass spectrometry approaches, including MALDI-TOF MS, tandem MS and Electron Transfer Dissociation coupled with Proton Transfer Reaction (ETD/PTR) top-down sequencing of N- and C-termini. Furthermore, the LSI-2a dimerization surface has also been investigated by a combination of gel filtration, electrophoretic techniques and homology modelling. Knowing the structure of small proteins inhibiting proteolytic enzymes is of general importance for understanding the defence mechanisms against degradation for their use in biological applications as well as for designing artificial inhibitors.
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, I-81100 Caserta, Italy.
Rachele
R
Tamburino
Valeria
V
Severino
Annamaria
A
Sandomenico
Menotti
M
Ruvo
Augusto
A
Parente
Angela
A
Chambery
Antimo
A
Di Maro
D000595:Amino Acid Sequence; D027807:Lathyrus / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012639:Seeds / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D010940:Plant Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors