A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

Abstract

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.

The xenograft antigen in complex with GS-1-B4lectin: crystallization and preliminary X-ray analysis

“…The GS-4 structure (PDB code 1gsl; Delbaere et al, 1993) produced the best results. Although the sequencing of GS-1-B 4 is in progress, a BLAST (Altschul et al, 1990) alignment of the N-terminal GS-1-B 4 sequence (Lamb & Goldstein, 1984) showed a 44% identity with that of GS-4. The initial cross-rotation search (DeLano & Bru È nger, 1995) gave a solution with a rotation function value of 0.0657 at 4 = 0.25 at 1 = 334, 2 = 78, 3 = 278 .…”

Lectin histochemistry of the normal human endometrium and endometrial adenocarcinoma.

“…West and Cope likewise noted the neuraminidaseengendered GS-I reactivity of endometrial epithelia (25) and results shown in their report were consistent with the present ones. GS-I is a tetramer and there are two types of subunit, A and B (14). Commercially available GS-I consist of A4 or B4, or might be any combination of A and B subunits.…”

Isolectins I-A and I-B of Griffonia(Bandeiraea) simplicifolia

“…Reverse Transcription-PCR and cDNA Cloning of G. simplicifolia GS I-A and GS I-B Isoforms-PCR was used to amplify the coding region of GS I-A and GS I-B isoforms using degenerate primers that were designed with regard to N-terminal peptides, previously characterized for both isoforms (11), and conserved internal peptide regions deduced from the multialignment of the whole protein family. Total RNA was obtained by grinding G. simplicifolia seeds in liquid nitrogen followed by an extraction using TRIzol reagent (Sigma Chemical Co.) according to the manufacturer's instructions.…”

“…Sequencing of the 25 N-terminal amino acids of the A and B subunits demonstrated 4 different residues and tryptic peptide mapping showed that ϳ30% of the peptides are common to both lectins (11). Thus, the GS I lectins appear to be excellent models to study the molecular basis for recognition of Gal versus GalNAc monosaccharides.…”