Semisynthetic Lectin–4-Dimethylaminopyridine Conjugates for Labeling and Profiling Glycoproteins on Live Cell Surfaces

Abstract

Glycoproteins on cell surfaces play important roles in biological processes, including cell-cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both in vitro and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells.

Antagonizing the Androgen Receptor with a Biomimetic Acyltransferase

“…Recently, Hamachi et al developed ligand-tether DMAP catalysts that can work in conjunction with excess thioester labeling reagents to label proteins of interest. 10 Although proximity directed DMAP catalysis was quite selective for cell surface proteins where competing nucleophiles are limited, intracellular protein targets showed only partial selectivity even after optimization of reagent reactivity. 11 These DMAP based nucleophilic catalysts are reminiscent of the prior work by Appella et al who previously demonstrated that the thiosalicylamide MT-1 can serve as a nucleophilic acyl transfer catalyst capable of inactivating HIV Gag by endogenous cellular metabolites as a source of reactive acetate.…”

“…In the presence of excess added labeling reagent, these ligand-tethered catalysts can label selectively cell surface proteins by directing the transfer of reactive thioesters. 10 They have also used ligand-DMAP conjugates to target intracellular protein targets but with only partial selectivity. 11 …”

Enantioselectivity and catalysis improvements of Pseudomonas cepacia lipase with Tyr and Asp modification

“…34,35 As a powerful tool, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can be used to solve the problem. 40,41 Here MALDI-TOF-MS was used to verify that Tyr and Asp residues were reactive towards NAI, I 3 − , EDC and EDA. [36][37][38][39] Recently, MALDI-TOF-MS was applied to determine the modification sites of congII and the affinity binding sites of boronic acid-decorated lectins.…”

Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications

“…From a standpoint of organic chemistry, it is also crucial to expand the repertoire of organic reactions that can be used for traceless affinity labeling of proteins in biological conditions. For instance, we have recently developed two new techniques, the affinity-guided 4-dimethylaminopyridine (DMAP) (AGD) chemistry [43][44][45] and the ligand-directed acyl imidazole (LDAI) chemistry [46,47], both of which allow rapid selective native protein labeling. Other promising ligand-directed chemistries have also recently been emerging [48][49][50][51][52].…”