title: Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry.
abstract: A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W102) in the activity of the lectin.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Gnanesh
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Padmanabh
authors.initials: P
authors.lastname: Mishra
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Vellareddy
authors.initials: V
authors.lastname: Anantharam
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000373:Agglutinins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001974:Bromosuccinimide / Q000737:chemistry; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D029776:Luffa / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000373:Agglutinins; D009844:Oligosaccharides; D014312:Trisaccharides; C041161:chitotriose; D014364:Tryptophan; D001974:Bromosuccinimide
keywords: chemical modification; chitin; de novo sequencing; mass spectrometry; phloem lectin