Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells
Yu C, Crispin M, Sonnen AF, Harvey DJ, Chang VT, Evans EJ, Scanlan CN, Stuart DI, Gilbert RJ, Davis SJ. (2011), Acta Crystallogr Sect F Struct Biol Cryst Commun. 67, 785-9
Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.
Key figure: Deglycosylation and crystallization of the apo CTLA-4 homodimer
(a) Coomassie-stained SDS–polyacrylamide gel run under reducing conditions showing undigested (−) and Endo H-digested (+) CTLA-4ex expressed in wild-type and mutant CHO cells with and without glycan-processing inhibitors. Samples in the left and right lanes marked with (+) were Endo H-digested for 1 and 3 h, respectively, in order to confirm that after 1 h the reaction had proceeded to completion. Sample 1, CHO-K1 cells only; sample 2, CHO-K1 cells with 1.5 mM NB-DNJ; sample 3, CHO-K1 cells with 10 µM kifunensine; sample 4, CHO Lec18.104.22.168 cells only; sample 5, CHO Lec22.214.171.124 cells with 0.5 mM NB-DNJ. In (b) crystals were grown in 25%(w/v) polyethylene glycol 1500 in 0.1 M sodium propionate/sodium cacodylate/Bis-Tris propane buffer pH 6.0 (Molecular Dimensions). These crystals were ∼100 × 100 × 100 µm in size. The crystal shown in (c) was grown in 0.2 M ammonium acetate, 25%(w/v) polyethylene glycol 1500, 0.1 M Bis-Tris pH 5.5 (Hampton Research). This crystal was ∼100 × 200 × 100 µm in size.