Báo cáo khoa học: Highly site-selective stability increases by glycosylation of dihydrofolate reductase ppt

Glycosylation stabilizes many proteins against ther-mal denaturation [14–17], whereas the removal of car-bohydrates from naturally glycosylated proteins can lead to decreased thermal sta

dihydrofolate reductase

Lai-Hock Tey1, E Joel Loveridge1, Richard S Swanwick1,*, Sabine L Flitsch2and Rudolf K Allemann1

1 School of Chemistry, Cardiff University, UK

2 School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, UK

Introduction

Post-translational glycosylation is one of the most

abundant forms of covalent protein modification in

eukaryotic cells and plays an important role in

deter-mining the properties of proteins, affecting many

molecular processes in vivo [1–5] There are two main

types of protein glycosylation: N-glycosylation, in

which the oligosaccharide is attached to an asparagine

side chain, and O-glycosylation, in which it is attached

to the side chain of serine or threonine residues [4]

Surface glycoproteins act as markers for inter- and

intracellular communication, and glycosylation has

been shown to affect a number of protein properties

such as structure, dynamics, stability and catalytic

activity [6–14]

Glycosylation stabilizes many proteins against ther-mal denaturation [14–17], whereas the removal of car-bohydrates from naturally glycosylated proteins can lead to decreased thermal stability and an increased tendency towards protein aggregation [18–20] Some studies have shown that glycans reduce the rate of unfolding but do not affect refolding of denatured pro-teins, leading to the conclusion that glycans preferen-tially bind to the folded protein and therefore stabilize

it [20–24] Others have also shown that folding is pro-moted in the presence of glycans [18,25,26], suggesting that the effects are protein specific Notably, many proteins show considerable increases in thermostability when in solution with high concentrations of sugars or

Keywords

enzyme; glycosylation; kinetics;

mutagenesis; stability

Correspondence

R K Allemann, School of Chemistry, Cardiff

University, Main Building, Park Place, Cardiff

CF10 3AT, UK

Fax: +44 29 2087 4030

Tel: +44 29 2087 9014

E-mail: allemannrk@cf.ac.uk

*Present address

Department of Life Sciences, Imperial

College, London, UK

(Received 10 January 2010, revised 26

February 2010, accepted 2 March 2010)

doi:10.1111/j.1742-4658.2010.07634.x

Post-translational glycosylation is one of the most abundant forms of cova-lent protein modification in eukaryotic cells It plays an important role in determining the properties of proteins, and stabilizes many proteins against thermal denaturation Protein glycosylation may establish a surface micro-environment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols We have used site-directed mutagen-esis to introduce a series of unique cysteine residues into a cysteine-free double mutant (DM, C85A⁄ C152S) of dihydrofolate reductase from Escherichia coli (EcDHFR) The resulting triple mutants, DM-N18C, DM-R52C, DM-D87C and DM-D132C EcDHFR, were alkylated with glucose, N-acetylglucosamine, lactose and maltotriose iodoacetamides We found little effect on catalysis or stability in three cases However, when DM-D87C EcDHFR is glycosylated, stability is increased by between 1.5 and 2.6 kcalÆmol)1 in a sugar-dependent manner D87 is found in a hinge region of EcDHFR that loses structure early in the thermal denaturation process, whereas the other glycosylation sites are found in regions involved

in the later stages of temperature-induced unfolding Glycosylation at this site may improve the stability of EcDHFR by protecting a region of the enzyme that is particularly prone to denaturation

Abbreviations

DM, double mutant; EcDHFR, Escherichia coli dihydrofolate reductase.

other polyols [27] Protein glycosylation may therefore

establish a surface microenvironment that resembles

that of unglycosylated proteins in such solutions

Several methods have been described for the

genera-tion of neoglycoproteins via site-selective glycosylagenera-tion

of proteins using chemical modification of

biotechno-logically produced proteins [2,3,28–31] One such

approach combines site-directed mutagenesis, to

intro-duce unique cysteine residues at the required sites, and

a highly flexible but selective chemical derivatization strategy (Scheme 1) in which reaction of the free thiol group of a cysteine residue with a synthetic glycosyl iodoacetamide produces a stable linkage between the protein and the carbohydrate [30] which resembles that found in native glycosylation of asparagines [32–34]

We have previously used this approach to study of the effect of site-specific glycosylation on the physical and chemical properties of the naturally nonglycosylated

Scheme 1 Strategy used for the synthesis

of highly purified glycosylated Escherichia coli dihydrofolate reductase triple mutants [30] A unique cysteine residue on the pro-tein is first reacted with a glycosyl iodoace-tamide (glucose is used as an example here); unalkylated proteins are biotinylated

by reaction with 2-((biotinoyl)amino)ethyl methanethiosulfonate Treatment with resin-bound avidin removes the biotinylated protein from solution, leaving highly purified neoglycoprotein.

enzyme dihydrofolate reductase

(5,6,7,8-tetrahydro-folate : NADP+ oxidoreductase, EC 1.5.1.3) from

Escherichia coli (EcDHFR) [14] EcDHFR catalyses

the stereospecific reduction of 7,8-dihydrofolate to

(6S)-5,6,7,8-tetrahydrofolate using NADPH as a

cofac-tor [35], and is therefore responsible for maintaining

the tetrahydrofolate pool within the cell EcDHFR is a

monomeric enzyme made up of eight b-strands, four

a helices and a number of important loop regions; it is

typically divided into three subdomains, the

adenosine-binding domain, the substrate-adenosine-binding domain and the

loop domain (Fig 1) [36] Our previous study was

based on a cysteine-free C85A⁄ C152S double mutant

of EcDHFR (DM EcDHFR), which has similar

fold-ing, stability and kinetic properties to the wild-type

enzyme (WT EcDHFR) [37] Cysteine residues were

introduced at two sites and the effect of glycosylation

at these sites was studied [14] Substitution of a

cysteine residue at position 87 (to form DM-D87C

EcDHFR) caused a loss in thermostability of the

protein that was reversed on glycosylation, whereas

DM-E120C EcDHFR had similar thermostability to the native enzyme and subsequent glycosylation led to

a smaller increase in melting temperature than that observed at position 87 [14] The kinetic parameters of the steady-state reaction catalysed by EcDHFR were not significantly affected by mutation and subsequent glycosylation at either position [14] This difference in response to glycosylation at the two sites was intrigu-ing and prompted further study Here, we describe the effect of glycosylation at three further sites on EcDHFR and report the kinetic properties, thermal stability and chemical stability at room temperature of the resulting glycoproteins The sites chosen were N18,

on the catalytically important M20 loop, R52, respon-sible for binding the glutamate tail of the substrate, and D132, ‘behind’ the active site at the end of the FG loop (Fig 1) Our results suggest that the local envi-ronment of the protein is critically important in deter-mining the effect of the glycosyl chain on protein unfolding

Results

Preparation of glycosylated EcDHFR mutants Double and triple mutants of EcDHFR were prepared using standard molecular biology techniques and the proteins expressed, purified, glycosylated and further purified as described previously [30] Prior to glycosyl-ation, all proteins were > 95% pure as judged by SDS–PAGE Glycosylation was confirmed by tryptic digestion followed by MALDI-TOF MS (Fig S1)

Ligand binding and kinetics of glycosylated EcDHFR Quenching of the enzyme fluorescence at 340 nm was used to determine the equilibrium dissociation con-stants of enzyme–NADPH and enzyme–folate com-plexes All five mutants have KDvalues similar to WT EcDHFR for both NADPH and folate (supporting information) The largest change was seen for DM-R52C with folate, where a threefold loss of affin-ity was seen In addition, no significant differences between the kinetic parameters of the five mutants and those of the wild-type protein were observed in either the steady state or pre-steady state, nor were there any reliable trends in the values on glycosylation (support-ing information)

Stability of glycosylated EcDHFR The far-UV CD spectra of the EcDHFR double and triple mutants and of the glycosylated triple mutants

Fig 1 Structure of Escherichia coli dihydrofolate reductase

(PBD 1RA2) [36] showing the position of the five residues mutated

to cysteine for this study The two views are rotated 180 about

the z-axis relative to each other The adenosine-binding domain

(ABD), substrate-binding domain (SBD), loop domain (LD) and

spe-cific loops mentioned in the discussion are indicated The enzyme

is shown as a cartoon representation; residues of interest and

ligands are shown as sticks H2F, 7,8-dihydrofolate.

were all similar to those of the wild-type enzyme,

indi-cating that neither the mutations per se nor

glycosyla-tion had an effect on the secondary structure of the

proteins large enough to be detectable by CD

spectros-copy (supporting information) Thermal denaturation

of WT EcDHFR and the five mutants was reversible

from 80 to 20C, and the melting temperatures of all

proteins except DM-D87C EcDHFR were similar

(Table 1) It has previously been shown that DM

EcDHFR has similar stability to the WT protein [37]

As previously reported, the thermal denaturation

tem-perature of DM-D87C EcDHFR is almost 10C lower

than that of WT EcDHFR, even though there is no

significant difference in the secondary structure of the

two proteins, and stability is restored by glycosylation

[14] The change in stability is because of the glycan

rather than the acetamide linkage [14] Stability of the

glycosylated mutant proteins was also determined

using equilibrium urea titrations monitored by

trypto-phan fluorescence emission (Table 2 and supporting

information) Mirroring the thermal stability results,

DM EcDHFR and three of the four triple mutants

showed little change in resistance to urea denaturation,

although DM-D87C EcDHFR showed a considerably lower free energy of unfolding, indicating a signifi-cantly lower stability The free energy of unfolding of DM-D87C EcDHFR was increased by glycosylation, although the other mutants were unaffected The sta-bility of glycosylated DM-D87C EcDHFRs increased with the length of the glycosyl chain; monosaccharides caused a similar increase in free energy of unfolding as incubating the nonglycosylated enzyme in a 0.5 m solu-tion of maltose, whereas larger sugars gave a more pronounced effect

Discussion

We have previously reported a large reduction in ther-mal stability for DM-D87C EcDHFR and its subse-quent ‘rescue’ by glycosylation [14] The same study showed a slight increase in thermal stability on glyco-sylation of DM-E120C EcDHFR Here we demon-strate that three further EcDHFR triple mutants show similar stability (against both temperature- and urea-induced denaturation) to the wild-type protein and that, in these cases, glycosylation does not improve

Table 1 Melting temperatures of EcDHFR, its mutants and their glycosylated forms Values were determined by CD spectroscopy using

10 l M enzyme in 5 m M potassium phosphate buffer (pH 7.0) DM, double mutant; EcDHFR, Escherichia coli dihydrofolate reductase; WT, wild-type, ND, not determined.

Glycan

Tm(C)

WT

EcDHFR-C85A ⁄

a Single measurement.

Table 2 Free energy of unfolding of Escherichia coli dihydrofolate (EcDHFR), its mutants and their glycosylated forms Values were deter-mined by fluorescence intensity measurement of urea-induced unfolding of 2 l M enzyme in 10 m M potassium phosphate buffer (pH 7.0).

DM, double mutant; WT, wild-type.

Glycan

DG (kcalÆmol)1)

WT

EcDHFR-C85A ⁄

stability (Table 1) In all cases except that of

DM-D87C EcDHFR, site-selective glycosylation had a

smaller effect on the thermal stability of the proteins

than the presence of 0.5 m maltose A similar trend

was observed for the stabilities of the proteins at

ambient temperature with respect to denaturation

induced by urea (Table 2) Notably, although the free

energy of unfolding of DM-D87C increased with

increasing glycan length, the thermal stability did not

show such a trend and was instead highest with the

monosaccharide N-acetylglucosamine Glycosylation of

DM-D87C EcDHFR with lactose or maltotriose

acetamides had a larger effect on the free energy of

unfolding than a 0.5 m solution of maltose

Interest-ingly, the changes in free energy of unfolding were

because of changes in the gradient of the urea

depen-dence of the free energy (supporting information),

rather than changes to the mid-point of the

urea-induced unfolding This suggests that glycosylation of

DM-D87C EcDHFR affects the cooperativity of the

unfolding transition rather than simply the resistance

to denaturants [38] The results presented here provide

further support [14] that, at least in the case of

dihy-drofolate reductase, increased stability through the

addition of glycans is because of highly site-specific

effects rather than nonspecific changes to the solvation

properties of the enzyme, suggesting that stabilization

of EcDHFR relies on specific interactions between the

protein and the glycan In the case of DM-D87C

EcDHFR, it appears that glycosylation increases the

effective concentration of sugar at a critical site to

more than that provided by a 0.5 m solution of

malt-ose In fact, the increase in melting temperature is

sim-ilar to that seen in a 1.5 m (50% w⁄ v) solution of

sucrose [39] By contrast, site-selective glycosylation of

the protein in regions unimportant for glycan-induced

stability would produce no benefit, as observed here

Inspection of the EcDHFR structure reveals no

fea-ture around position 87 that would be expected to

interact particularly favourably with glycans

Compu-tational [40,41] and experimental [42] work has

indi-cated that the ‘hinge’ region of the adenosine-binding

domain in which D87 is found unfolds very early in

the denaturation process, although others [43–45] have

suggested an alternative folding pathway in which this

region would be expected to unfold slightly later If

this region does lose structure early in the unfolding

process, this may explain why the stability of

EcDHFR is sensitive to glycosylation at this site –

regions more vulnerable to denaturation are likely to

benefit more from additional stabilizing interactions

N18, E120 and D132 are all found in the loop

domain, which retains structure until relatively late in

the thermal unfolding process [40,41], whereas R52 is formally located in the adenosine-binding domain but forms part of the substrate-binding pocket Sugars bound at position 52 are therefore more likely to interact with the relatively stable [40–42] substrate-binding domain (Fig 1) Hence glycosylation at these positions may not exert a similarly stabilizing effect as glycosylation at position 87

Both ligand binding and the kinetics of EcDHFR were remarkably robust to the mutations made and subsequent glycosylation The most notable difference

in KD values was observed for DM-R52C EcDHFR with folate, although this is still only an approximately threefold increase R52 forms part of the binding site for the glutamate tail of the folate ligand, whereas N18 forms part of the M20 loop, which closes over NADPH after it enters the active site (Fig 1) It has previously been shown that reacting DM-N18C and DM-E17C mutants with bulky groups has little effect

on their kinetics or ligand binding relative to EcDHFR [46,47] E120 and D132 are both located on the FG loop, important because of its interactions with the M20 loop that controls progression through the cata-lytic cycle [36,48] Mutation of glycine 121 to bulkier residues causes a sharp decrease in catalytic activity, and a reduction in the affinity for NADPH [49,50] However, this is likely to be because of global struc-tural changes observed for the G121V mutant [40], which disrupt the ability of the EcDHFR : NADPH complex (and the reactive Michaelis complex) to form its native ‘closed’ conformation [48] Changes at posi-tion 120, where the side chain is exposed to solvent, would not be expected to produce so pronounced an effect The absence of large effects on catalysis pro-vides further evidence that mutation and subsequent glycosylation do not produce significant changes in the global structure of the enzyme, but that stability of EcDHFR may be affected by binding of sugars to specific sites on the enzyme

In conclusion, our previous study suggested that the thermal stability of proteins can be increased signifi-cantly by the attachment of even relatively small car-bohydrates, rather than the larger oligosaccharides typically found in nature [14] We now report a similar effect on chemical stability at room temperature, and add that the local environment of the protein appears

to be critically important in determining the effect of bound oligosaccharides The large oligosaccharides observed in nature may allow greater coverage of a number of discrete, critical points of stabilization from

a single glycosylation site, rather than being simply caused by blanket coverage of large regions of the protein surface Alternatively, increases in stability

because of protein–carbohydrate interactions close to

the attachment site may be coupled to other functions

(such as molecular recognition) at the ends of the

gly-can chain Our results suggest that glycosylation at

position 87 of EcDHFR may improve its stability

by protecting a region that is particularly prone to

denaturation

Experimental procedures

Protein preparation

EcDHFR triple mutants were generated by site-directed

mutagenesis of DNA encoding DM EcDHFR in the same

way as for DM-E120C EcDHFR [30] Mutagenic primers

GTGG-3¢ (N18C), 5¢-CTGGGAATCAATCGGTTGCCCG

TTGCCAGGAC-3¢ (R52C), 5¢-GCGGCGGCGGGTTGC

GTACCAGAAATCATGG-3¢ (D87C) and 5¢-CCGGATTA

CGAGCCGGATTGCTGGGAATCGG-3¢ (D132C) The

cysteine codons are underlined All unglycosylated proteins

were purified by methotrexate affinity and anion-exchange

chromatography as described previously [40] Purified triple

mutants were subsequently glycosylated with glucose,

N-acetylglucosamine, lactose and maltotriose acetamides

and further purified as described previously for DM-E120C

EcDHFR [30]

CD spectroscopy

Experiments were performed using an Applied

Photophys-ics (Leatherhead, UK) Chirascan spectrometer at a protein

concentration of 10 lm in 5 mm potassium phosphate

buf-fer (pH 7.0) Spectra were acquired between 200 and

280 nm To monitor thermal denaturation, spectra were

acquired between 20 and 80C using a temperature

gradi-ent of 0.4CÆmin)1 Unfolding of the protein was

moni-tored at 222 nm and the melting temperature was taken as

the midpoint of the observed transition Thermal

denatur-ation measurements were performed in triplicate

Determination of free energy of unfolding

Equilibrium unfolding of the proteins and their

deriva-tized glycoforms was monitored in the presence of urea

by the fluorescence intensity at 345 nm and 20C using a

Perkin–Elmer (Beaconsfield, UK) LS55 Luminescence

spectrometer Urea solutions were prepared freshly for

each experiment and treated with AG 501-X8

ion-exchange resin (Bio-Rad, Hemel Hempstead, UK) The

protein concentration was maintained at 2 lm in 10 mm

potassium phosphate buffer (pH 7.0) containing 0.1 mm

EDTA, 0.1 mm dithiothreitol and the required

concentra-tion of urea All samples were incubated overnight at

room temperature prior to measurement Between 10 and

15 data points were acquired to adequately define the denaturation curve, and the free energy of unfolding was determined using the linear extrapolation method [38] All unfolding measurements were performed in triplicate

Ligand-binding experiments

Equilibrium dissociation constants (KD) of the protein com-plexes with folate and NADPH were measured at 20C by monitoring quenching of the intrinsic tryptophan fluores-cence as a function of ligand concentration using a Perkin– Elmer LS55 Luminescence spectrometer Folate was used in place of dihydrofolate because of its enhanced stability Protein concentrations were 0.05 or 0.5 lm (for titration with NADPH and folate, respectively) in 50 mm potassium phosphate buffer (pH 7.0) containing 50 mm NaCl, 0.1 mm EDTA and 0.1 mm dithiothreitol Ligand concentrations were 0.1–9.5 lm for NADPH and 1–125 lm for folate Dissociation constants were determined by fitting the nor-malized fluorescence intensities (F) data to the Langmuir isothermFFit= {1 + (KD⁄ [Ligand])n})1, where n = 1 (i.e

1 : 1 binding) gave the best fits

Enzyme kinetics

All kinetic measurements were performed in MTEN buffer (50 mm Mes, 25 mm Tris, 25 mm ethanolamine, 100 mm NaCl pH 7.0) at 20C Steady-state rates were measured spectrophotometrically by following the decrease in absor-bance at 340 nm during the reaction (e340, NADPH+DHF

= 11 800 m)1Æcm)1) The enzyme (10 lm) was incubated with NADPH (20 lm) for 15 min to avoid hysteresis [51] This enzyme–NADPH solution (5 lL) was added to 950 lL buffer and NADPH (1–100 lm final concentration) added The reaction was started by adding dihydrofolate (100 lm final concentration) Each experiment was performed in triplicate and the rates calculated from the linear fittings of the initial velocities KMNADPHand kcatwere determined by fitting the data to the Michaelis–Menten equation The KM value was not determined for dihydrofolate because of the lower stability of this compound

Pre-steady-state kinetic experiments were performed on

an Applied Photophysics stopped-flow spectrometer with 2.5 mL drive syringes EcDHFR (8 lm) was preincubated with NADPH (4 lm) for at least 15 min at 20C and the reaction initiated by rapidly mixing with an equal volume of dihydrofolate (100 lm) The dead time of the experiment was < 2 ms The reaction was monitored by fluorescence energy transfer using a 400 nm cut-off filter and excitation at 292 nm Rate constants were determined

by fitting the observed kinetic traces to single or double exponential decay using software provided with the instrument

The financial support from the UK’s Biotechnology

and Biological Sciences Research Council (BBSRC)

through grants 6⁄ B15285 (SLF, RSS and RKA) and

BB⁄ E008380 ⁄ 1 (RKA and EJL) and from Cardiff

Uni-versity (studentship to L-HT)

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Supporting information

The following supplementary material is available: Fig S1 MALDI-TOF MS following trypsin digestion

of EcDHFR triple mutants

Fig S2 CD spectra at 20 C and thermal melting curves of WT EcDHFR and DM EcDHFR

Fig S3 Urea denaturation curves and free energy of unfolding for WT EcDHFR and DM EcDHFR Fig S4 Binding curves for NADPH and folate with

WT EcDHFR and DM EcDHFR

Fig S5 CD spectra at 20 C and thermal melting curves of EcDHFR triple mutants

Fig S6 Free energy of unfolding at 20 C for EcDHFR triple mutants

Table S1 Mean residue ellipticities at 222 nm.

Table S2 Midpoints of the urea-induced unfolding

transition

Table S3 Gradients of the free energy of unfolding

plots

Table S4 Dissociation constants for NADPH

Table S5 Dissociation constants for folate

Table S6 Hydride transfer rate constants

Table S7 Steady-state turnover rates

Table S8 Michaelis constants

Table S9 kcat⁄ KM

This supplementary material can be found in the online version of this article

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