Quantitative Effects of O-Linked Glycans on Protein Folding

Jul 26, 2017 - One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantag...
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Quantitative Effects of O-linked Glycans on Protein Folding Patrick K Chaffey, Xiaoyang Guan, Xinfeng Wang, Yuan Ruan, Yaohao Li, Suzannah G Miller, Amy H Tran, Theo N Koelsch, Lomax F Pass, and Zhongping Tan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00483 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Quantitative Effects of O-linked Glycans on Protein Folding Patrick K. Chaffey,‡ Xiaoyang Guan,‡ Xinfeng Wang, Yuan Ruan, Yaohao Li, Suzannah G. Miller, Amy H. Tran, Theo N. Koelsch, Lomax F. Pass, Zhongping Tan* Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303, United States ABSTRACT: Protein O-glycosylation is a diverse, common, and important post-translational modification of both proteins inside the cell and those that are secreted or membrane bound. Much work has shown that O-glycosylation can alter the structure, function, and physical properties of the proteins to which it is attached. One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantage of synthetic, homogeneous O-glycopeptides to show that certain glycosylation patterns have an intrinsic effect, independent of any cellular folding machinery, on the folding pathway of a model O-glycoprotein, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A. The strongest effect, a six-fold increase in overall folding rate, was observed when a single O-mannose was the glycan, and the glycosylation site was near the N-terminus of the peptide sequence. We were also able to show that glycosylation patterns affected the kinetics of each step in unique ways, which may help to explain the observations made here. This work is a first step towards quantitative understanding of how O-glycosylation might control, through intrinsic means, the folding of O-glycoproteins. Such an understanding is expected to facilitate future investigations into the effects of glycosylation on more biological processes related to protein folding.

INTRODUCTION Proteins are important across many fields as basic and critical components of living systems, tools that answer scientific questions, industrial enzymes that produce and modify compounds on a commodity scale, drug targets and, increasingly, therapeutics in their own right.1-5 Since a proper threedimensional shape, or fold, is essential for a protein’s function, a better understanding of how to improve the folding process is important for any application of proteins, their production, or their biochemical and biological characterization.6-9 It has been shown that glycosylation contributes favorably to protein folding.10-12 Protein glycosylation most commonly comes in two varieties: N-linked glycosylation where the glycan units are attached to the protein backbone via the nitrogen atom of an Asn side chain and O-linked glycosylation where the attachment point is the oxygen of a Ser or Thr side chain.13-15 Through many years of studies, the role of Nglycosylation as a check-point in protein folding and quality control has been well established.16 In addition, N-linked glycans, mainly the core structure, have been shown to accelerate folding, and stabilize the folded, native state of the glycoprotein conjugate independent of any outside chaperone interactions.17 N-glycans can also promote the formation of correct disulfide bonds and secondary structural elements.18 These effects may be accomplished by the combined action of specific contacts between the glycan and protein, like hydrogen bonds and CH-π interactions, as well as non-specific effects due to their steric bulk and large hydration spheres.11 This wealth of knowledge of protein N-glycosylation was facilitated by developments in the controlled preparation of relatively homogeneous N-glycosylated proteins for study17-19 and, in turn, has provided valuable insight into strategies to glycoengineer proteins.20, 21

O-Glycosylation has also long been associated with protein folding.13, 22, 23 However, despite of many decades of research, the correlation between folding and protein O-glycosylation has not been sufficiently investigated. Chief among the difficulties for obtaining such knowledge is the lack of homogeneous samples for investigation. Unlike N-glycosylation, the term “O-glycosylation” is actually a blanket term that describes many unique types of glycosylation, including the addition of N-acetylgalactosamine (GalNAc), Nacetylglucosamine (GlcNAc), mannose (Man), galactose (Gal), fucose (Fuc), glucose (Glc), or xylose (Xyl) to Ser or Thr.13, 24, 25 More complicatedly, in contrast to N-glycosylation, which is initiated in the endoplasmic reticulum (ER), the initiation sites of different types of O-glycosylation vary. Depending on the first sugar unit that is attached to protein side chain, O-glycosylation can take place either in the ER or the Golgi apparatus, and either before or after protein folding.13 Many different enzymes are responsible for the initial Oglycosylation reaction and the complex regulatory networks that control the outer saccharides of the glycans. Most of them have not been well characterized. Further complicating matters is the fact that the most common types of O-glycosylation (OGalNAcylation and O-mannosylation) have no defined consensus sequence for their introduction.25 Together, these factors render it challenging, if not impossible, to using molecular biology and biochemical approaches similar to those developed for N-glycoproteins to prepare structurally-defined Oglycopeptides or glycoproteins for folding study.26, 27 To better understand and apply protein glycosylation, it is necessary to examine the detailed effects of O-linked glycans on protein properties. To address the challenges associated with such studies, we have taken advantage of recent advances in chemical synthesis19, 28-33 and developed a chemical glycobiology approach to analyze changes caused by the O-

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glycosylation of proteins.34, 35 This approach can potentially be applied to molecules with up to ~200 amino acid residues,36 an upper limit which conveniently covers many soluble proteins.37, 38 Compared to recombinant expression methods, chemical synthesis is a more precise and flexible way to generate molecules with small and predetermined variations in structures, thus allowing the quantitative evaluation of the effects of different sugar moieties at different sites. Here, we applied a chemical glycobiology approach to carry out a detailed study on the effects of O-glycosylation on protein folding in vitro, centering on the changes caused by varied O-mannosylation patterns and differences between mannose and other O-glycan types. Of the many types of Oglycosylation, O-mannosylation is the most abundant in fungi and the second most common in the mammalian nervous system.13, 25 There are also several pieces of evidence that tie Omannosylation to protein folding pathways. For example, blocking O-mannosylation leads to aggregated and mis-folded proteins accumulating in the ER39 and blocking Nglycosylation can lead to an upregulation of O-mannosylation which might be a compensatory adjustment by cells to limit the mis-folding of proteins normally aided by Nglycosylation.40 Additionally, the biosynthesis process for Omannoyslation is strikingly similar to that of N-glycosylation, as both are initiated in the ER by transferring initial glycans via dolichol derivatives. After initiation, the first O-linked mannose residue is extended in the Golgi apparatus to give a variety of complex structures.23 This information hints that under certain conditions, O-mannosylation may play a similar role to N-glycosylation and have a strong connection to protein folding.39, 41 Therefore, it is of great interest to first focus the study on this type of O-glycosylation.

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teins: contains three glycosylation sites, two at the N-terminus and one in the middle. It forms two disulfide bonds and folds into a well-defined three-dimensional structure. These features make the CBM ideal molecule for the early study of the role played by O-glycans in folding.

MATERIALS AND METHODS Materials. All commercial reagents and solvents were used as received. Unless otherwise noted, all reactions were performed under argon and purifications were performed under air atmosphere at room temperature. Automated peptide synthesis was performed on an Applied Biosystems Pioneer continuous flow peptide synthesizer. All LC-MS analyses were performed using a Waters AcquityTM Ultra Performance LC system equipped with Acquity UPLC® BEH 300 C4, 1.7µm, 2.1 x 100 mm column at flow rates of 0.3 and 0.5 mL/min. The mobile phase for LC-MS analysis was a mixture of H2O (0.1% formic acid, v/v) and acetonitrile (0.1% formic acid, v/v). A Waters Synapt G2 HDMS (q-TOF) system was used for the mass spectrometric analysis. Design and chemical synthesis CBM variants. Previous research has demonstrated that combining the data from a wide variety of glyco-variants can provide a clear view of not only the influence of protein glycosylation but also the structural factors behind that influence 18, 35, 43. Therefore, we selected 25 CBM glycoforms for the present study, which can be divided into three series for a systematic study of three different features of CBM O-glycosylation. The first series (Fig. 2, 2-10) was meant to investigate both the site-specific and glycan-size dependent consequences of O-mannosylation. The second series (Fig. 2, 11-16) looked at the synergistic effects of O-mannoses at multiple sites. The final series (Fig. 2, 1725) probed how the stereochemistry of the glycan units and linkage along with the amino acid side chains near a glycan site alter the effects of O-glycosylation on folding. The unglycosylated CBM peptide 1 was also included as a control.

Figure 1. The NMR structure of the Family 1 CBM with three Olinked mono-mannose residues at Thr1, Ser3, and Ser14 sites.42 The O-linked glycans, the glycosylated amino acids and the disulfide bonds between Cys8 and 25 and Cys19 and 35 are shown in sticks. The oxygen atoms in the stick representations are in red color, carbon in green, and sulfur in gold.

Like most of our previous studies, we used a model molecule, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A,34, 35, 42 to investigate the effects of O-mannosylation. The CBM is a small, structurally independent domain of only 36 amino acids. Its small size is an advantage since it allows us to prepare large collections of glycoforms in a reasonable amount of time and makes it much easier to gather quantitative observations (Fig. 1). Despite its small size, it has the key features of typical glycopro-

Figure 2. Amino acid sequences and glycosylation patterns of the CBM variants. The native disulfide bonds are indicated with solid

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black lines connecting the Cys residues. Glycans are indicated by R1, R2 and R3.

The synthesis of the unglycosylated and glycosylated CBM variants 1-25 was accomplished in a one-pot process following our previously developed procedures.34, 35 Fmoc protected amino acid (4.0 eq.), HATU (4.0 eq.) and DIEA (8.0 eq.) were used for the coupling steps. The amino acid derivative FmocSer(Ac4Glcα1)-OH, was synthesized by procedures based on previous work (Supporting Information).44-46 The deblock solution was a mixture of piperidine/DBU/DMF (2/2/100, v/v). Upon completion of automated synthesis on a 0.05 mmol scale, the peptide resin was cleaved with TFA/TIS/H2O (95/2.5/2.5, v/v, 10 mL) for 45 min at room temperature. The resin was filtered and washed with TFA/TIS/H2O (95/2.5/2.5, 10 mL). The filtrate was combined and concentrated by compressed air. The oily residue was precipitated with diethyl ether and centrifuged to give a white pellet. After ether was decanted, the solid was dissolved in 10 mL of MeCN/H2O (50/50, v/v,) and was lyophilized to dryness. Correct folding, disulfide bond formation, purity and identity of each variant were experimentally verified by circular dichroism (CD) spectroscopy, thermolysin digestion, and analytical ultraperformance liquid chromatography-mass spectroscopy (UPLC-MS) 34, 35. Measuring the overall folding rates of CBM variants. Overall rates of folding for each CBM variant were measured using a procedure analogous to that developed previously in our lab to produce fully folded CBM variants for later characterization and study.34, 35 Solid, lyophilized CBM peptide (3 mg) was dissolved in 180 µL hydrazine/H2O (5/95, v/v) and stirred for 30 minutes at room temperature. Hydrazine was quenched by addition of 360 µL hydrazine quench solution (AcOH/H2O, 5/95, v/v). Solution was then added to 12 mL of folding buffer (0.2 Trizma, 3 mM reduced glutathione, pH = 8.2) and allowed to stir at room temperature. During this time, at each time point (t = 0 min, 5 min, 15 min, 30 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min, 480 min), a sample (40 µL) was removed from the folding solution, quenched with 136 µL of TFA/H2O/MeCN (0.5/85/15, v/v), and stored at -70 oC until LC-MS analysis. This procedure was carried out in triplicate and results were averaged. LC-MS was carried out with a gradient of 20→40% acetonitrile in water on a C4 column. For each sample collected, all the mass spectrum scans were combined and the weighted mass average was calculated. The [M+3H]3+ peak was used for this purpose because it was the highest intensity peak observed in all samples. Weighted average mass is calculated by multiplying the intensity of each peak by the m/z value for that peak, summing up all these numbers and dividing by the sum total of the intensities of each peak. When no disulfide bond formation has taken place, this weighted average mass matches the expected molecular weight calculated by taking into account the natural isotopic distributions of each element in the CBM peptide absent any disulfide bonds. Similarly, once folding is completed, the weighted average mass calculated this way matches the expected molecular weight of the CBM peptide with both disulfide bonds. An example of this analysis is illustrated in the Supporting Information (Fig. S2) for the unglycosylated CBM variant. Plotting the change in mass (relative to the CBM peptide without disulfide bonds) against time gives a curve that reflects the oxidative folding process of each CBM variant, and

fitting this curve allows for the calculation of a rate constant of the overall oxidative folding for each variant. Since these curves were linear through the vast majority of the folding process for each peptide, linear fits of the data were used and the slope of the resulting lines were taken as the rate constants. Tracking each molecular species during folding. CBM folding involves the formation of at least three molecular species: the fully reduced (0S) and fully oxidized (2S) as well as intermediate species containing only one disulfide bond (1S). For the CBM variants that showed good chromatographic separation of these species during the folding reaction, we further examined the changes of each species during folding. Our analysis began with the assignment of the peak for each molecular species according to mass. An example of this analysis for glycosylated CBM variant 5 is shown in the Supporting Information (Fig. S4). Once identified by mass, the area under each peak was tracked for each molecular species for each time point collected. Plotting the percent of the total area under the curve each species represented gave the plots seen in Figures 4 and S5. This analysis was done using data from each of the three trials collected above for overall folding rate. Thus folding reactions were only performed once. Initial rate constants for each species were calculated based on the initial linear portion of each curve, and at least four points were used in each case.

Figure 3. Two representative folding trajectories monitored by mass spectrometry. The upper panel shows the folding of CBM

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variants with several sizes of O-mannosyl glycans at Thr1, the smallest of which led to significant changes in CBM folding rates. The bottom panel shows the folding of CBM variants with several sizes of O-mannosyl glycans at Ser14, none of which had a large effect on CBM folding.

RESULTS Impact of O-glycosylation on the overall folding rates of CBM variants. The process of protein folding is a complicated process.47 For proteins containing disulfide bonds, like CBM, it is a combination of two distinct, but interrelated processes: conformational folding of the protein sequence into secondary and tertiary structures and formation of disulfide bonds. Collectively, these processes are known as oxidative folding.48 Conformational shuffling is typically a fast process, while the disulfide bond formation steps are much slower.49-51 By taking advantage of the relatively slow kinetics in disulfide bond formation, methods that monitor oxidative folding have facilitated detailed characterizations of several disulfide containing proteins’ folding pathways and thus significantly advanced our understanding of the molecular basis of protein folding.47, 48 We opted here to also make use of disulfide-bond chemistry to compare the folding rates of different CBM variants in vitro.50, 52, 53 To elucidate the potential site- and size specific effects of O-mannosylation on the folding of CBM, nine monoglycosyl-

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ated variants, 2-10, were compared with the nonglycosylated CBM 1 on their folding rate constants (Fig. 3, 4). As previously reported, the folding rate of each variant was quantified based on the measurable mass difference between the partially and fully folded CBM and the unfolded peptide.54 Briefly, the folding reactions were initiated by dissolving each CBM peptide in a folding buffer of pH 8.2 containing 0.2 M Trizma and 3 mM reduced glutathione (GSH).55, 56 We tested several different combinations of reduced and oxidized glutathione (GSSG) for the buffer and found that using only the reduced glutathione gave the cleanest chromatographic separation of folding intermediates and the lowest amount of misfolded product (Supporting Information, Fig. S1). At different time intervals after initiation, small aliquots were removed from the folding reaction and quenched by adjusting the pH to 3.57 Because each new disulfide bond formed results in a loss of two hydrogen atoms, the average mass of the peptides in each sample could be used as a measure of the overall progress of oxidative folding at the time of aliquot collection.54, 58 Such aggregate measures of a protein’s folding pathway, which cannot specifically interrogate the individual partially folded intermediates, have a long history in the study of protein folding. Indeed, most of the early papers in the area used enzymatic activity as a measure of how folded the enzyme is under particular conditions.59, 60 Since the CBM domain has no enzymatic activity, and high-resolution mass spectroscopy has advanced significantly in the past fifty years, we chose to use

Figure 4. The relative overall folding rate constants of the synthetic CBM variants. These values were calculated by normalizing each folding rate constant to that of the unglycosylated CBM 1. All error bars reported are standard deviations (SDs) of data achieved from three separate trials. Numerical data can be found in the Supporting Information (Table S1). ** indicates p≤0.001, * indicates p≤0.05, two-tailed t-test. The structural feature of each isoform is implied by its name, i.e. CBMT1M1 representing the isoform containing a single mannose α-linked to Thr1, CBMT3M3+S3M3+S14M3 representing the isoform containing a tri-mannose α-linked to Thr1, Ser3, and Ser14, CBMS3ManNAcα representing the isoform containing a single N-acetylmannosamine α-linked to Ser3, and CBMY5A+S3(Manα) representing the isoform containing a Tyr-to-Ala mutation at position 5 and a single mannose α-linked to Ser3.

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the change in mass that accompanies a disulfide bond formation step to monitor the overall folding progress.47, 57, 61 The curves of the average mass as a function of time displayed nearly linear behavior during the majority of the folding reaction, and so the slopes of linear fits were used to calculate an observed rate constant for each variant (Fig. 3 and Supporting Information Fig. S3).62, 63 As shown in Figure 4, left, our data shows that addition of a single mannose at either Thr1 or Ser3 significantly increased the overall folding rate, while Ser14 had very little effect on the rate as compared to unglycosylated CBM (compare the rate constants of 2, 5 and 8 with 1). For each site, increasing the size of the linear mannose chains resulted in unchanged rate constant as compared to that of unglycosylated CBM (compare 3, 4, 6, 7, 9, and 10 with 1). To better understand the impact of O-glycosylation on the folding of CBM, we next conducted a study of the folding rates of CBM glycoforms with O-linked mannoses at multiple sites. As shown in Figure 4, middle, multiple mannose glycans on the CBM can result in slightly slower or unchanged folding rates, even when the glycans are at the same sites that individually increase the rate (compare 11-13, 15 and 16 with 1). Of the multiply glycosylated variants, the glycoform with a single mannose at each of the three glycosylation sites displayed an increase in the rate (compare 14 with 1). We next decided to investigate how structural details of the carbohydrate moiety contribute to its effect on the folding rate of CBM. For this, we focused on glycosylation at Ser3, which was shown to significantly alter the folding rate when the glycan was mono-mannose and had been revealed in our previous work to have the largest effect on several other physical properties of the peptide.34,35,66 Like our previous experiments, we compared the rate constants of several CBM variants with changes this time to the orientation of functional groups on the sugar ring.35 As shown in Figure 4, right, our results showed that these small changes significantly decreased the differences in the rate constants between the glycosylated variants and the unglycosylated control (compare 17-23 with 1). Of all the amide-containing carbohydrates, only Nacetylmannosamine (ManNAc), which is stereochemically most similar to Man, had a noticeable positive impact on the folding rate (compare 17-19 with 1). Comparing the α- and βlinked Glc and Gal containing glycoforms showed that neither the carbohydrate identity nor the anomeric linkage stereochemistry in these two groups of mono-saccharides had any large influence on the rate (compare 20-23 with 1). Our previous work indicating that both Gln2 and Tyr5 played important roles in stabilizing the CBM structure, hence we were also interested in how these residues might alter the folding rate. As shown in Figure 4, right, mutating either Gln2 or Tyr5 to Ala abolished the accelerating effect of mono-mannose at Ser3, resulting in similar folding rates as the unglycosylated CBM (compare 24, 25 with 5 and 1). Impact of O-glycosylation on the changes of individual molecular species involved in the folding process. Comparing the overall folding rates provided evidence that most Oglycosylation patterns only minimally affect this process, but specific patterns can drastically accelerate oxidative folding. However, the information obtained from this kind of analysis is necessarily limited and a clearer picture of the folding process for each CBM variant is desirable. It is certainly possible, given the evidence uncovered from previous studies, that the glycans might bias the folding pathway to favor formation of

one disulfide bond or direct formation of two disulfide bonds in a glycan-dependent manner.47, 48 We therefore set out to characterize the effects of O-glycosylation on different molecular species, mainly focusing on the initial rates of formation of 1S and 2S, peak time of 1S, and initial rates of the unfolded starting material, 0S (Fig. 5).

Figure 5. Changes in the relative abundances of the 0S ( ), 1S ( ), and 2S ( ) molecular species that contain 0, 1, and 2 disulfide bonds, respectively, during the folding of select CBM variants: CBMS3M1 5, CBMS14M3 10, and CBMS3Glcα 20, each representing a different folding pattern. All error bars reported are SDs of data achieved from three separate trials.

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For a more detailed analysis of folding, we needed a way to track each molecular species individually. Fortunately, we found that, for analogs 5-7, 10-15, 17-23, and 25, the UPLCMS gave modestly resolved separation of the different species observed during folding (Supporting Information Fig. S4). By mass, peaks could be identified for 0S, 1S, and 2S forms. Once the peaks for these species were identified, the area under each peak could be used as a measure of the amount of each species in the folding mixture. Comparing the area of each peak to the total area under the curve for the trace gives a measure of the relative concentration of those species.64 Since we could resolve these peaks by retention time, use their mass as an indication of their identity, and quantify their concentrations based on peak areas, we were able to track the formation and disappearance of each species, and calculate their initial rates of formation or disappearance, and peak times. The resulting plots produced from the detailed analysis of three representative examples, CBMS3M1 5, CBMS14M3 10, and CBMS3Glcα 20, are shown in Figure 5 (Supporting Information contains all the data obtained this way, Fig. S5). As seen in the results, each of the CBM variants showed some similarities in the folding process. At the beginning, the samples mainly contain pure 0S for each analog. As time passes, the fraction of 0S falls while the fraction of 2S increases. The fraction of 1S rises initially before reaching a peak and falling. By comparing the 2S products with purified, folded peptides for which proper disulfide bond connectivity has been verified in our previous studies,35, 65 we can conclude that the 2S products observed in these experiments are correctly folded. Interestingly, the presence of cross-disulfide species consisting of a molecule of glutathione linked to the CBM, which is expected to occur given the fact that glutathione was used in the folding buffer,66 was not seen on any of the UPLC-MS traces. This suggests that these intermediates are very short-lived and react

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quickly to form either a new disulfide bond in the forward direction or reduced starting material in the backward direction. However, despite these similarities, there are significant differences in the reactions of different molecular species, probably reflecting differences in the rates of formation and reaction of this intermediate between differently glycosylated CBM analogs. This can also be illustrated by the data of analogs CBMS3M1 5, CBMS14M3 10, and CBMS3Glcα 20. As shown in Figure 5, the disappearance of the 0S species in CBMS3M1 5 follows an exponential decay, rapidly halving the initial concentration within the first hour. This sharp decrease is coupled to a sharp increase in the fraction of 1S species observed, which quickly peaks at around 40% of the total at the end of the first hour of folding. The 2S species increases at a notably slower pace and follows a mostly linear trend upwards until about three hours into the reaction when about three quarters of the CBMS3M1 5 is in the 2S form. After eight hours, the reaction is essentially complete with negligible amounts of 0S and 1S species, and almost complete conversion to the 2S species. In contrast, CBMS14M3 10 shows a consistently linear rate of disappearance of the 0S species. The fraction of the 1S species increases steadily as well, but notably at a much slower pace than that of CBMS3M1 5. As with CBMS3M1 5, the 1S species of CBMS14M3 10 peaks at around 40% of the total, but takes around four hours to reach this level. Another sharp contrast between CBMS14M3 10 and CBMS3M1 5, is the build-up of 2S in CBMS14M3 10. It was unobservable during the first hour of folding, appeared as a very low fraction of the total after two hours, and then steadily increased in a linear fashion until reaction completion. The folding of CBMS14M3 10, like CBMS3M1 5, was essentially complete after eight hours, although there was a small amount of 1S species remaining in the final sample. CBM

Figure 6. Initial rate constant for each species (0S, 1S and 2S) observed in the oxidative folding reactions. Rate constants derived from fitting the initial linear portion of each curve over at least 4 data points. Only CBM variants with good chromatographic separation on UPLC for each of the three species were used in this analysis. Rate of disappearance of 0S is in white and is negative. Rates of formation of 1S and 2S are in grey and black, respectively, and are both positive. Bars represent the average rates from three trials and error bars come from the standard deviation of those three trials. Numerical data can be found in the Supporting Information (Table S2).

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CBMS3Glcα 20 presents yet another variation. The 0S species disappears with an initially high rate before leveling out to be largely linear. The 2S species is formed following an exponential growth pattern, increasing in rate throughout the eight hours measured during the experiment. Unlike both CBMS3M1 5 and CBMS14M3 10, the 1S species peaks at only around 20% of the sample and reaches this peak after 2 hours of folding. After eight hours, the folding of CBMS3Glcα 20 is far less complete than either CBMS3M1 5 or CBMS14M3 10, with only around three-quarters of the total CBM being in the 2S form and a significant amount remaining in the totally reduced 0S form. To quantify the differences between each of CBM variants, we determined the initial reaction rates for each of the molecules. Since the concentrations of intermediates and products are negligible in the initial rates region, the initial rates may more accurately reflect the effects of glycosylation. In total, we were able to achieve a detailed analysis of the folding of 17 CBM glycoforms. As shown in Figure 6, our results revealed that glycosylation of Ser3 with a mannose monomer (CBMS3M1 5) resulted in rates of formation for both 1S and 2S species, and also a rate of disappearance for 0S, that are much higher than most of the other analogs for which data was available. While CBMS3M2 6 had similarly large initial rates for formation of 1S species, its rate of formation of the final 2S product was very low; and overall, the rate of 0S disappearance was lower than that of CBMS3M1 5. It seems that the mannose dimer of CBMS3M2 6 slows mainly the rate of formation of 2S species, while the mannose trimers of CBMS3M3 7 and CBMS14M3 10 seem to slow both the formation of the 1S intermediate and the 2S product. Multiply glycosylated CBMS1M1+S3M1+S14M1 14, which was observed to have an increase in the observed overall folding rate constant, also showed relatively large initial rates for all three species monitored, although not nearly as large as those of 5. Additionally, our results suggest that mono-mannose has a uniquely significant ability to increase the initial rates of formation for 1S and 2S species. All the other monomeric glycans tested showed much lower initial rates for both the formation of the 1S and 2S, as well as the rate of disappearance of 0S.

DISCUSSION Protein glycosylation is an extremely prevalent posttranslational modification, especially among secreted and membrane proteins.14 It can have important effects, both extrinsic and intrinsic.11, 67 Because a very specific consensus sequence makes obtaining homogeneous N-glycosylated proteins fairly convenient, most previous studies have focused on deciphering the effects of N-glycans.10, 68, 69 From these studies, it has been shown that the first sugar unit, GlcNAc, which is directly attached to the protein, confers almost the entire accelerating effect of N-glycans on protein folding.17 Although the exact physical and chemical explanations for these phenomena are not known with absolute certainty, there is considerable evidence that contacts, including hydrogen bonds, hydrophobic interactions, and CH-π interactions, between the core carbohydrates of the N-glycan structure and the proximal amino acids in the protein are the root cause of these observations.17, 21 Although it has thus far received less attention, Oglycosylation is also very common on proteins.70 Whether and how protein O-glycosylation participates in protein folding

and quality control is poorly understood. Recent results show that O-linked mannoses are involved in ER protein quality control, but it is not clear if they also affect other closely related folding properties or what structural features determine these effects.23, 41, 71 As detailed here, through a systematic study of O-glycosylated CBM variants, we were able to quantify our understanding of protein O-glycosylation’s contributions in the realm of oxidative folding. From those results, it was observed that only the first mannose residue is responsible for O-mannose glycans’ acceleration of folding rate in the CBM. This finding, together with the fact that Omannosylation biosynthesis involves the transfer of monomannose to selected amino acid residues in ER with further diversification only in the Golgi apparatus,23, 72 suggests that mono-mannoses added in the ER may to some extent be analogous to N-glycans by acting to intrinsically accelerate correct folding of proteins in vivo. These results also match well with recent work showing that O-fucosylation of thrombospondin type 1 repeats (TSRs) likely represents a novel ER-based quality control mechanism for ensuring TSR-containing proteins achieve their proper disulfide pairing patterns.73 Many naturally-occurring glycoproteins contain more than one glycosylation site. Therefore, it is also essential to establish the differences of different sites in regulating protein folding and the potential of interplay between these sites. Such analysis has rarely been performed so far, in large part due to the difficulty of selectively targeting only particular glycosylation sites using glycosyltransferases without changing protein amino acid sequences.20 The synthetic methods used here to prepare glycosylated CBM variants nicely side-step this issue and allowed us to show that effects of O-glycosylation are site-specific and there is no synergistic enhancement of the folding rate conferred by different glycosylation sites. In particular, our results highlight the importance of the N-terminal sites, both Thr1 and Ser3, in accelerating the folding of CBM over the more central Ser14 site. Protein folding is a complex process involving many different types of interactions.74 In order to develop a better understanding of the effects of O-glycosylation on folding, it is necessary to get some insights into the molecular determinants of such effects. Our results clearly showed that of a suite of monomeric glycans, only O-linked mannose is optimal for accelerating the folding of CBM. Additionally, mutating specific amino acids that are adjacent to a glycosylation site decreases or abolishes the effects of mannoses. Such dependence suggests that the peptide sequence of CBM may be evolutionarily adjusted to satisfy O-mannosylation and the interplay between the mono-mannose and amino acids at the N-terminus that is necessary for the effects induced by the mono-mannosylation at Thr1 and Ser3. An important question is whether glycosylation patterns with overall rate-accelerating effects simultaneously increase the rates of all the steps in the folding process or just some steps. By providing more information about the detailed effects of glycosylation on protein folding, our study revealed an interesting, but not unexpected correlation. From the data presented in Figures 4 and 6, it seems that glycosylation that increases the overall folding rate also increases the formation of both 1S intermediates and 2S products. In summary, by systematically comparing the properties of differently glycosylated CBM variants in vitro, we found that O-mannosylation has a strong site- and size-specific influence

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on the folding of the CBM. Our results clearly show that only the attachment of a mono-mannose to the two N-terminal glycosylation sites can significantly increase the overall folding rate of CBM. The greatest increase occurring at Thr 1, with an observed folding rate more than 5 times faster than that of unglycosylated CBM. Also noteworthy is the fact that monomeric O-mannose glycans are added in the ER during early protein synthesis, and these were seen here to accelerate the rate of protein folding. Meanwhile larger mannosyl glycans are generated after oxidative folding in the Golgi and were seen here to have a negligible effect on folding. Thus, the effects of O-mannosylation on protein folding seem to vary in a manner that mirrors the biosynthetic pathways of Omannosylated glycoproteins. Our results also support that many factors contribute to the effects of glycosylation and small changes in either glycan structures or amino acid sequences can lead to large differences in the ability of glycosylation to have an effect on protein folding. Furthermore, our data suggest that glycosylation has different effects on the rates of each step in the folding process, although only an acceleration of the rates of all steps will lead to an observed increase in the overall rate. Taken together, this study illustrates that O-glycosylation can significantly affect the folding process of proteins and suggests a role for O-linked glycans in expediting glycoprotein biosynthesis. The generality and implications of these findings need to be explored through detailed in vitro studies of other glycoproteins paired with rigorous in vivo work. Such efforts are currently being pursued and we hope they will answer the many remaining questions.

ASSOCIATED CONTENT Supporting Information. Experimental procedures, spectroscopic and analytical data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

ORCID: 0000-0002-9302-150X Notes The authors declare no competing financial interest.

Author Contributions All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

ACKNOWLEDGMENT We would like to thank the University of Colorado Boulder (Start-up fund), the NSF CAREER Award (Grant number: CHE1454925) for their support during the course of this study.

ABBREVIATIONS 0S, fully reduced CBM; 1S, CBM species containing one disulfide bond; 2S, fully oxidized CBM; CBM, carbohydrate-binding module; Fuc, fucose; Gal, galactose; GalNAc, N-

acetylgalactosamine; Glc, glucose; GlcNAc, Nacetylglucosamine; Man, mannose; TrCel7A, Trichoderma reesei cellulose; Xyl, xylose.

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