Protein Glycosylation, Sweet to Crystal Growth? - ACS Publications

In protein crystal growth, glycosylation is often considered an annoyance because of its impact on homogeneity and surface entropy. However, in our ex...
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CRYSTAL GROWTH & DESIGN

Protein Glycosylation, Sweet to Crystal Growth?† Jeroen R. Mesters* and Rolf Hilgenfeld Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, UniVersity of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

2007 VOL. 7, NO. 11 2251–2253

ReceiVed July 23, 2007; ReVised Manuscript ReceiVed September 18, 2007

ABSTRACT: In protein crystallization, post-translational modifications are often considered an annoyance. They are expected to introduce microheterogeneity into the protein and, in addition, in the case of the larger chemical modifications such as glycosylation, to increase surface entropy. On the one hand, these phenomena are said to be bad for crystal growth. On the other hand, many of these modifications are vital for proper folding and functioning of proteins. Over the past few years, we successfully crystallized several different glycoproteins, obtained by heterologous expression or directly isolated from their natural source. The crystallization of these proteins turned out to be fairly straightforward, and moreover, the carbohydrates were frequently found to be involved in forming (critical) intermolecular contacts. Opposite widespread opinions, an unbiased approach in crystallizing glycoproteins should prevail initially. Finally, several methodologies are listed that could be tried as a last resort. The list of known chemical modifications of amino acid residues in a polypeptide is terribly extensive, ranging from a rather simple phosphorylation or methylation to the more complex lipidation or glycosylation.1 In protein crystal growth, co/post-translational modifications however are almost always considered an annoyance. They are said to introduce significant microheterogeneity into the protein sample and, in the case of large chemical modifications, to seriously enhance surface entropy. Although both phenomena are thought to be bad for crystal growth, many chemical modifications are vital for a proper folding and/or functioning of proteins. Glycosylation is considered particularly challenging concerning microheterogeneity and surface entropy. In eukaryotic proteins, carbohydrates are connected to the amide nitrogen of asparagine (N-glycosidic bond) and to the hydroxyl group of threonine, serine, and hydroxy-lysine (O-glycosidic bond). These protein modifications take place in the endoplasmatic reticulum (ER) and Golgi apparatus: The first steps in N-glycosylation occur cotranslationally in the ER and require dolichol-phosphate (lipid anchor), whereas O-glycosylation normally takes place post-translationally in the Golgi.2–4 Interestingly, protein O-glycosylation can also occur outside the Golgi, in the cytosol and nuclear space.5 In recent years, it became clear that protein glycosylation is a trait of all kingdoms of life.6 In bacteria, both periplasmic and cell-surface proteins can become glycosylated. The bacterial N-glycosylation pathway is surprisingly similar to the eukaryotic one in that a lipid-linked, oligosaccharide is preassembled in the cytosol, translocated, and finally transferred to the protein.7 Bacterial glycosylation patterns exhibit an unequalled diversity and, in Gram-negative species (i.e., pathogens of clinical importance), glycoproteins are often associated with virulence.8,9 Unfortunate, and regardless of the ubiquitous consensussequence Asn-X-Ser/Thr (with X not Pro) in N-glycosylation,10 prokaryotic expression systems do not produce N-glycosylated eukaryotic proteins. This is mainly because of the cotranslational character of N-glycosylation in eukaryotes as opposed to the post-translation character in bacteria: Unfolded eukaryotic proteins are better substrates for bacterial glycosyltransferases.11 † Part of the special issue (vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Que´bec, Canada, August 16–20, 2006 (preconference August 13–16, 2006). * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +49-451-5004068. Phone: +49-451-5004070.

Not surprisingly, cotranslational N-glycosylation has an immediate effect on protein folding.12 One of the key steps in structural genomics (i.e., crystal growth) is the production of suitable amounts of correctly folded, soluble protein. Because of the inadequacies of bacterial expression systems, eukaryotic expression systems have been widely used and adapted13–16 to produce glycoprotein samples for structural biology.17–23 The developments of these approaches were to a certain extent driven by the belief that glycoproteins are notoriously difficult to crystallize. Over the past few years, we successfully crystallized a number of different glycosylated proteins, obtained by either heterologous expression or directly isolated from their natural (plant) source. Our own experiences suggest to initially approach the crystallization of glycoproteins in an unbiased manner. Crystallization of Glycosylated Proteins Reports in the literature of successful crystallization after deglycosylation apparently fuel the widespread opinion, especially among novices, that glycosylated proteins are very difficult to crystallize.24–26 Notwithstanding, over the past few years, we successfully crystallized a number of different glycosylated proteins (discussed below), obtained by either heterologous expression or directly isolated from their natural (plant) source. The enzyme alliinase catalyzes the cleavage of alliin, leading to precursors of therapeutically active sulfur compounds from garlic. The homodimeric glycoprotein could be isolated from fresh, homegrown garlic in ultrahigh purity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis: Isoelectric focusing slab polyacrylamide gel electrophoresis (IEF–PAGE) examination however showed a ladder of protein bands, revealing serious microheterogeneity.27 Nonetheless, four different crystal forms could be obtained, all of which diffracted X-rays to better than 4.0 Å resolution. IEF–PAGE analysis of dissolved crystals revealed the same amount of microheterogeneity as present in the original proteinstock solution. One of these crystal forms diffracted X-rays up to 1.53 Å resolution. The quaternary structure illustrated that the carbohydrates are clearly involved in homodimer formation.28 PAP-S, isolated from the seeds of Chinese pokeweed (Phytolacca acinosa), belongs to the family of the type-1 ribosome inactivating proteins (i.e., site-specific depurination of the

10.1021/cg7006843 CCC: $37.00  2007 American Chemical Society Published on Web 10/24/2007

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Figure 1. Overall structure of the homodimeric GCPII in complex with an inhibitor. Seven out of ten glycosylation sites (sugars shown as colored sticks) were visible in the electron density maps (PDB code 2JBJ). The water channels within a macromolecular crystal lattice are often able to accommodate the very flexible carbohydrate chains. The glycosyl residues attached to Asn638/Asn638′ are involved in homodimer formation (encircled). Molecular diagram was drawn using the PyMOL molecular graphics suite (DeLano Scientific LLC, Palo Alto).

R-sarcin/ricin loop in rRNA). Crystals of PAP-S were successfully grown from a heterogeneous mixture of two isozymes of 29 and 30 kDa, respectively.29 An polyacrylamide gel electrophoresis SDS–PAGE analysis of dissolved crystals however showed only one single protein band of 30 kDa. The crystals diffracted X-rays to 1.70 Å resolution. Intriguingly, in the refined structure, one carbohydrate was found to be exclusively responsible for the crystal lattice formation across a 2-fold crystallographic axis.29 Human glutamate carboxypeptidase II is a large, membraneanchored glycoprotein that occurs in the central nervous system as well as in human prostate. Natural substrates of this zincmetalloenzyme are N-acetyl-L-aspartyl-L-glutamic acid and folate polyglutamate. The fully glycosylated protein (amino acid residues 44–750), harbouring 10 glycosylation sites, was produced using Drosophila Schneider cells.30 For five of these sites, the asparagine to alanine mutation caused a greater than 10fold drop in catalytic activity.31 The glycoprotein in complex with an inhibitor crystallized without difficulty and the crystals diffracted X-rays to 2.0 Å resolution.32,33 One of the carbohydrate chains (attached to Asn638; Figure 1) is clearly involved in homodimer formation: Mutation of Asn638, or Thr640, reduced the catalytic activity to below 2%. All in all, the crystallization of these glycosylated proteins turned out to be rather straightforward and, moreover, the carbohydrates were found to be involved in forming critical intermolecular contacts. Our overall success rate in crystallizing glycoproteins stands at ∼50–60%, which is neither better nor worse than using ordinary proteins. Thus, normally, neither the total carbohydrate content nor its heterogeneity appears to interfere with crystal growth.34 Not unexpected, the water channels within a macromolecular crystal lattice are able to cope with (i.e., stow away), the very flexible carbohydrate chains.35 Generally unexpected, crystals of glycosylated proteins can diffract X-rays up to atomic resolution (1.12 Å).36 Furthermore, due to crystal contacts, glycan structures can be well defined in

electron-density maps, revealing protein-linked carbohydrate chains of up to 10 sugars (1.75 Å resolution).37 Carbohydrate-Reduced or Carbohydrateless Several strategies have been developed for producing carbohydrate-reduced or carbohydrateless glycoproteins for structural investigations. Regularly, eukaryotic expression systems have been used, and the glycoproteins were enzymatically deglycosylated afterwards.24,25,34 This technique has been refined further in that glycosidase inhibitors such as N-butyldeoxynojirimycin or glycosylation processing inhibitors such as kifunensine and swainsonine were added during cell culture.38–41 Even tunicamycin could be of use, although the yields are generally lowered.42,43 Nevertheless, the postproduction enzymatic Ndeglycosylation is quite expensive,44 challenging, and often does not eradicate carbohydrate heterogeneity completely. O-Glycans can be treated with enzymes such as neuraminidase or O-glycanase.45 Deglycosylation protocols that, for obvious reasons, leave the innermost sugar(s) intact are to be preferred.29 To bypass difficulties, methods that achieve a more uniform, carbohydrate-reduced glycosylation could be tried, for example, expression in genetically engineered human embryonic kidney cells (HEK293S derived cells), genetically engineered Pichia pastoris cells, or Leishmania tarentolae cells.20,21,46 Occasionally, a simple bacterial expression system was used for the production of correctly folded and fully active, carbohydrateless glycoprotein. Most probably, this was only possible in cases where the carbohydrates have no effect on protein folding and activity. Conclusions In our experience, glycosylation does not a priori hinder crystallization (i.e., is sweet to crystal growth).47 Searching the Protein Data Bank (RSCB PDB) reveals that many hundreds of deposited structures carry carbohydrate modifications.48 Actually, the number of structures of successfully crystallized

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glycosylated proteins clearly outnumbers the cases in which crystals were exclusively obtained after (complete) deglycosylation. Many reports demonstrate that glycosylation is important for structural integrity and, moreover, can contribute extensively to crystal lattice formation.29 These observations underpin our judgment that, in general, neither the total carbohydrate content nor its heterogeneity appears to interfere with crystal formation.27,34 Of course, the diffraction limit and diffraction quality may occasionally suffer. Since the overall success rate in crystallizing glycoproteins is neither better nor worse than using ordinary proteins, we suggest to initially approach the crystallization of glycoproteins in an unbiased manner. If a glycoprotein fails to crystallize, aspects other than the carbohydrates are to blame: Many factors contribute to successful crystal growth, and maybe there was just one single silver bullet missing.47,49 Only if the unbiased approach fails completely, specific inhibitors followed by enzymatic deglycosylation or, even better, expression in customized HEK293S/Pichia pastoris/Leishmania tarentolae could be tried. Acknowledgment. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, Grant No. Me 2741/ 1-2) and, in part, by the “OptiCryst” project of the European Commission (Contract No. LSH-2005-037793) as well as the Schleswig-Holstein Innovation Fund. R.H. thanks the Fonds der Chemischen Industrie for continuous support.

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