Abstract
Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N-glycans’ characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.
Highlights
Lectins are proteins that bind to carbohydrates, either free or bound to cell membranes as part of glycoproteins, glycolipids, or polysaccharides
Production was scaled in a 7-L bench top fermenter, where a final yield of 316 ± 0.27 mg recombinant lectin/L of culture supernatant was obtained using colony 10 as the pre-culture. rTBL-1 was purified by nickel affinity chromatography, and fractions were collected after elution with 200 mM imidazole
A single copy insertion of a TBL-1 coding sequence into the P. pastoris genome leads to the high-performance production of rTBL-1 lectin (316 mg/mL culture)
Summary
Lectins are proteins that bind to carbohydrates, either free or bound to cell membranes as part of glycoproteins, glycolipids, or polysaccharides. Some of the most common tumor-associated glycan changes described to date include (1) increased β1-4 branched tetra-antennary N-glycans [4,5]; (2) α2-6 sialylation on the outer poly-N-acetyllactosamines (LacNAc) of N-glycans [6]; (3) increase of α1-6 fucosylation [7,8]; (4) overexpression of mucins and truncated O-glycans [9]; (5) altered expression of blood groups and sialyl-Lewis antigens [10]; (6) increase in bisecting GlcNAc [11]; (7) overexpression of hyaluronan [12,13]; and (8) increased β1-6 branching of N-glycans [4,5] The latter is one of the most important glycan modifications in colorectal cancer cells. It is derived from an upregulation of the N-acetylglucosaminyltransferase V (MGAT5) by oncogenic transcription factors from the RAS–RAF–MAPK signaling pathways [4], which are highly associated with cancer metastasis [11]
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