Glycoprotein structural genomics: solving the glycosylation problem

Abstract

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Figure 1

Manipulation of the Mammalian N-Linked Glycosylation Pathway in HEK293 Cells (A) A section of the glycosylation pathway is illustrated. (B–D) MALDI-TOF analysis of the glycans of the target glycoprotein (s19A) expressed in (B) HEK293T cells in the presence of the α-mannosidase I (MI) inhibitor, kifunensine, at 5 μM, resulting in the addition of oligomannose-type N-glycans, Man_5–9GlcNAc₂; (C) ricin-resistant HEK293S cells devoid of GnTI activity, resulting in predominantly Man₅GIcNAc₂N-glycans; and (D) HEK293T cells in the presence of the α-mannosidase II (MII) inhibitor, swainsonine, at 20 μM, resulting in the addition of hybrid-type glycans. For MALDI-TOF MS, glycans were released directly from SDS-PAGE gel bands by overnight PNGase F digestion. Monosaccharide constituents are represented as follows: open diamonds, Gal; closed diamonds, GaINAc; open squares, Glc; closed squares, GlcNAc; open circles, Man; stars, sialic acid; dotted diamonds, Fuc.

Figure 2

Endo H Digestion of s19A Produced in HEK293 Cells under Various Conditions (A–C) SDS-PAGE gels, run under reducing conditions, of endo H- or PNGase F-treated s19A expressed in (A) GnTI-deficient HEK293S cells, (B) HEK293T cells cultured with 5 μM kifunensine, and (C) HEK293T cells cultured with 20 μM swainsonine. In each case, 5 μg of purified s19A was treated at 37°C with 1 kU or 0.25 kU of endo H at the indicated pH, or at 37°C with 0.5 kU of PNGase F at pH 7.4, for 6 hr. For the endo H digests, identity of the products indicates that the digestions have gone to completion. (D) Crystals of endo H-treated sRPTPμ expressed in GnTI-deficient HEK293S cells (left panel) and endo H-treated s19A expressed in HEK293T cells in the presence of swainsonine (right panel). See Supplemental Data for the endo H-sensitivity and SDS-PAGE analysis of sRPTPμ. Data have been collected in space groups C2 and P2₁2₁2₁ for RPTPμ and s19A, respectively (see Experimental Procedures).