The enzyme-binding region of human GM2-activator protein

The enzyme-binding region of human GM2-activatorproteinMichaela Wendeler1, Norbert Werth1, Timm Maier2, Guenter Schwarzmann1, Thomas Kolter1,Maike Schoeniger1, Daniel Hoffmann3, Thorsten Lemm1, Wolfram Saenger2 and Konrad Sandhoff1

1 Kekule-Institut fur Organische Chemie und Biochemie der Universitat Bonn, Germany

2 Institut fur Chemie, Abt. Kristallographie, Freie Universitat Berlin, Germany

3 Forschungszentrum caesar, Bonn, Germany

Keywords

GM2-activator; ganglioside degradation;

b-hexosaminidase A; lipid transfer; lysosome

Correspondence

K. Sandhoff, Kekule-Institut fur Organische

Chemie und Biochemie der Universitat

Bonn, Gerhard-Domagk-Str. 1, D-53121

Bonn, Germany

Fax: +49 228 73 7778

Tel: +49 228 73 5346

E-mail: [email protected]

(Received 8 November 2005, revised 21

December 2005, accepted 28 December

2005)

doi:10.1111/j.1742-4658.2006.05126.x

The GM2-activator protein (GM2AP) is an essential cofactor for the lyso-

somal degradation of ganglioside GM2 by b-hexosaminidase A (HexA). It

mediates the interaction between the water-soluble exohydrolase and its

membrane-embedded glycolipid substrate at the lipid–water interface.

Functional deficiencies in this protein result in a fatal neurological storage

disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this

cofactor’s mode of action and identify the surface region of GM2AP

responsible for binding to HexA, we designed several variant forms of this

protein and evaluated the consequences of these mutations for lipid- and

enzyme-binding properties using a variety of biophysical and functional

studies. The point mutants D113K, M117V and E123K showed a drastic-

ally decreased capacity to stimulate HexA-catalysed GM2 degradation.

However, surface plasmon resonance (SPR) spectroscopy showed that the

binding of these variants to immobilized lipid bilayers and their ability to

solubilize lipids from anionic vesicles were the same as for the wild-type

protein. In addition, a fluorescence resonance energy transfer (FRET)-

based assay system showed that these variants had the same capacity as

wild-type GM2AP for intervesicular lipid transfer from donor to acceptor

liposomes. The concentration-dependent effect of these variants on hydro-

lysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-

sulfo-b-d-glucopyranoside (MUGS) indicated a weakened association with

the enzyme’s a subunit. This identifies the protein region affected by these

mutations, the single short a helix of GM2AP, as the major determinant

for the interaction with the enzyme. These results further confirm that the

function of GM2AP is not restricted to a biological detergent that simply

disrupts the membrane structure or lifts the substrate out of the lipid plane.

In contrast, our data argue in favour of the critical importance of distinct

activator–hexosaminidase interactions for GM2 degradation, and corrobor-

ate the view that the activator ⁄ lipid complex represents the true substrate

for the degrading enzyme.

Abbreviations

BMP, bis(monoacylglycero)phosphate; FRET, fluorescence resonance energy transfer; GM1, ganglioside GM1; GM2, ganglioside GM2;

GM2AP, GM2-activator protein; GSLs, glycosphingolipids; HexA, b-hexosaminidase A; HexB: b-hexosaminidase B; LUV, large unilamellar

vesicle; MUG, 4-methylumbelliferyl-2-acetamido-2-deoxy-b-D-glucopyranoside; MUGS, 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-b-

D-glucopyranoside; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; PtdCho, phosphatidylcholine; PE, dioleoyl glycero phosphoryl ethanolamine;

SAP, sphingolipid activator protein; SPR, surface plasmon resonance.

982 FEBS Journal 273 (2006) 982–991 ª 2006 The Authors Journal compilation ª 2006 FEBS

Glycosphingolipids (GSLs) are characteristic compo-

nents of eukaryotic plasma membranes. Over the last

20 years, their highly complex degradation pathway in

lysosomes and the metabolic diseases associated with

inherited defects in this pathway have been intensively

investigated [1]. In the case of GSLs with rather short

oligosaccharide head-groups of four or fewer sugar resi-

dues, these membrane-embedded substrates are not suf-

ficiently accessible for the degradation by water-soluble

enzymes, and the exohydrolases need the assistance of

small glycoprotein cofactors, the sphingolipid activator

proteins (SAPs) [2]. For hydrolytic conversion of gan-

glioside GM2 catalysed by b-hexosaminidase A (HexA,

EC 3.2.1.52), the GM2-activator protein is required

[3,4].

Three different isoforms of human lysosomal b-hexo-saminidases are known: b-hexosaminidase A (HexA),

the heterodimer of the noncovalently linked a chainand b chain, and the homodimeric isoenzymes b-hex-osaminidase B (HexB, bb) and b-hexosaminidase S

(aa). Despite the a and b subunits being 60% identical

in their primary structure and functionally very similar,

they show distinct specificity [5]. Only HexA is able to

degrade the physiologically most important substrate,

ganglioside GM2, at significant rates in the presence of

the GM2 activator. Defects in any of the three genes

encoding the polypeptides involved in GM2 degrada-

tion, the a and b subunit of HexA or the GM2AP,

result in the accumulation of nondegraded glycolipids

within the lysosomal compartment and the develop-

ment of severe neurodegenerative storage diseases

known as GM2-gangliosidoses [4,6,7].

In its mature form present in the lysosomes, the

GM2 activator is a small glycoprotein of 162 amino

acids [8]. It is known to bind avidly to a variety of ani-

onic lipids in vitro [9], to be able to extract several gly-

cosphingolipid monomers from micelles or liposomes

and to transport them as soluble 1 : 1 complexes

between donor and acceptor membranes [10]. The

membrane activity of GM2AP was found to depend

critically on several physiological parameters, most

notably a lateral pressure of the lipid bilayer below

15–25 mNÆm)1 [11] and the presence of anionic lipids,

in particular bis(monoacylglycero)phosphate (BMP)

[12]. Recently, a novel function of GM2AP in the con-

text of glycolipid antigen presentation via CD1d was

identified [13].

For the function of GM2AP in the lysosomal GM2

hydrolysis, the term ‘liftase’ was coined: it recognizes

the lipid substrate within the membrane plane and lifts

it out of the lipid bilayer, thereby presenting it to the

water-soluble enzyme for degradation [14]. In addition,

it has been suggested that GM2AP modifies the

conformation of the trisaccharide unit of ganglioside

GM2, thus facilitating the enzymatic hydrolysis of the

terminal N-acetyl-d-galactosamine (GalNAc) residue

[15]. Additional protein–protein interactions between

GM2AP and HexA were implied in the catalysis of

GM2 degradation [16,17]. Several lines of evidence

indicate that GM2AP interacts with the a subunit of

HexA, but the presence of the b subunit enhances

binding [5,16,18,19]. The crystal structure of mature

GM2AP expressed in Escherichia coli [20], as well as

that of lipid complexes of GM2AP [21,22], revealed a

novel protein fold, denoted b cup, which consists of an

eight-stranded antiparallel b-pleated sheet forming a

spacious hydrophobic cavity, as well as several surface

loops and a single short a helix. The dimensions of the

central pocket are such that it can accommodate the

ceramide tail of GM2. Using photoaffinity labelling,

we were able to establish that the most flexible of the

surface loops, the chain segment V153–L163 constitutes

the part of the activator protein that directly interacts

with the ganglioside substrate [23]. (The amino acid

numbering used here refers to the complete, prepro,

form of GM2AP, including residues 1–31 which are

removed by proteolytic processing. Please note that

Protein Data Bank entry 1G13 describing the crystal

structure of mature GM2AP expressed in E. coli, and

also Wright et al. [21], differ from this nomenclature;

they assign the number 1 to S32, which represents the

N-terminus of mature GM2AP.) This interaction might

be crucial for stabilization of the position of the glycoli-

pid within the spacious cavity, thereby ensuring the

correct orientation of the tetrasaccharide head-group

with respect to the degrading enzyme’s active site.

The goal of this study was to elucidate the interac-

tion between cofactor and enzyme and to develop a

comprehensive model for the GM2 degradation pro-

ceeding at a phase frontier on intralysosomal mem-

branes. Previously, attempts to identify the lipid- and

enzyme-binding region of GM2AP were hampered

because assay systems for GM2AP, which measure its

stimulatory potential for the degradation of GM2 by

HexA, reflect simultaneously both the interaction of

GM2AP with GM2 and the interaction of the

GM2AP ⁄GM2 complex with the enzyme. To delineate

in detail the separate effect of the introduced muta-

tions on the lipid- and the enzyme-binding of this

cofactor, we combined a variety of functional and bio-

physical analyses.

Crystallographic analysis of the homodimeric iso-

enzyme HexB [24,25] and subsequent comparative

modelling studies of heterodimeric HexA [25] suggested

that the dimer interface of the enzyme forms the dock-

ing site for the GM2AP ⁄GM2 complex. Furthermore,

M. Wendeler et al. Enzyme-binding region of GM2-activator protein

FEBS Journal 273 (2006) 982–991 ª 2006 The Authors Journal compilation ª 2006 FEBS 983

based on the crystal structures of the enzyme [24,25]

and of GM2AP [20–22], computational modelling and

theoretical docking studies identified the single short

ahelix of GM2AP as the region most likely to be in

direct contact with the enzyme. We therefore expressed

a series of site-directed mutants of GM2AP with amino

acid exchanges in this region of the protein. Only vari-

ants with conformations identical to the wild-type

(WT) GM2AP, as judged by CD spectroscopy, were

considered. To study the stimulatory potential of

GM2AP variants for ganglioside GM2 degradation by

HexA, conventional micellar assays, as well as a deter-

gent-free, liposomal assay system [12], were employed.

The influence on the hydrolysis of the synthetic sub-

strate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-

b-d-glucopyranoside (MUGS), which is degraded by

the a subunit of HexA and which hydrolysis is inhibited

by GM2AP [5], allowed probing the variant activator’s

binding to the enzyme. To study the membrane activity

and lipid-binding properties, SPR spectroscopy with

immobilized lipid bilayers was performed. Finally, a

recently developed assay based on fluorescence reson-

ance energy transfer (FRET) [26] enabled us to observe

in real-time the ability for intervesicular lipid transfer

from donor to acceptor vesicles.

This allowed identification of the a-helical region of

GM2AP as the major determinant in interacting with

the enzyme. Our data further confirm conclusively the

critical importance of distinct protein–protein interac-

tions in GM2 degradation and corroborate the view

that the activator ⁄ glycolipid complex represents the

true substrate for the degrading enzyme.

Results

To identify the protein region of GM2AP that inter-

acts with HexA in the lysosomal degradation of gan-

glioside GM2, we performed site-directed mutagenesis

of GM2AP and evaluated the biophysical and func-

tional properties of the variant proteins.

Initial homology modelling of HexA based on the

recently published crystal structure of human HexB

[25], and subsequent theoretical docking studies of the

GM2AP structure [20,21] suggested that the a helix of

GM2AP plays a major role in binding to the enzyme.

In this helix, comprising amino acids F111 to P120,

every fourth residue points in the same direction and

the side chains of D113 and M117 were found to be

oriented away from the GM2AP core towards the

putative interface with the enzyme. The first residue

following the GM2AP helix, which likewise points

towards this suggested interaction region, was E123.

The long and charged side chain of glutamate is

known to be particularly well suited for protein–pro-

tein interactions. In contrast, the residues between the

end of the helix and E123 (P120, T121, G122) most

likely serve a structural role only, allowing the loop to

adopt a bent conformation.

We therefore introduced the following mutations in

this region of the protein: D113K, D113A, D113Y,

M117V, E123K, E123A and E123Y. All variant pro-

tein forms were readily produced in the insect cell ⁄baculovirus expression vector system (BEVS) and puri-

fied to homogeneity by cation exchange and sub-

sequent Ni-NTA chromatography. Protein yields of

GM2AP mutants were in the range 4–7 mg purified

protein per litre of expression supernatant, compared

with 7–9 mg of the WT protein.

To monitor any possible disturbances in the protein

fold upon amino acid exchange, we subjected all

protein variants to UV circular dichroism (CD) spectro-

scopy (Fig. 1). Although secondary structure pre-

dictions suggested identical conformations for the

above-described GM2AP variants, it was found that

the point mutants D113A, D113Y, E123A and E123Y

showed altered CD spectra. In contrast, the mutations

M117V, D113K and E123K resulted in CD spectra

identical to that of the WT protein. Only these vari-

ants of GM2AP were examined in all further studies.

To assess the potential of the variant GM2AP forms

for the stimulation of HexA catalysed hydrolysis of

GM2, two established assays, involving either micellar

or liposomal glycolipid substrate, were performed. As

shown in Fig. 2, the mutant forms of GM2AP exam-

ined here showed a drastically decreased ability to

stimulate the hydrolysis of GM2 by HexA. In the mi-

cellar system (Fig. 2A), the point mutant M117V dis-

played 25–26%, the variant D113K 4–5% and E123K

3–4% of the WT capacity to stimulate the enzymatic

degradation of GM2. In the liposomal system (Fig. 2B),

which we introduced to mimic more closely the in vivo

reaction conditions on the surface of intralysosomal

vesicles and membrane structures [12], these results were

confirmed and the differences between WT protein and

mutants were even more pronounced. In this system,

the variant M117V showed 7–8% of the WT activity,

whereas the mutants D113K and E123K displayed

almost a complete loss of activity.

To assess the membrane activity and lipid-binding

behaviour of the GM2AP variants, their interaction

with immobilized lipid bilayers was measured using

SPR spectroscopy in a Biacore instrument (Fig. 3). All

mutant protein forms showed binding to the same

extent as the WT protein. In the presence of the ani-

onic lipid BMP, which is known to occur in inner

membranes of the acidic compartments and which

Enzyme-binding region of GM2-activator protein M. Wendeler et al.


stimulates membrane activity of GM2AP [12], all

mutants showed a comparable pronounced decrease in

the SPR signal during the subsequent injection of pro-

tein-free buffer. This response has been interpreted as

the solubilization of lipids from the surface of immobi-

Fig. 2. Stimulation of HexA-catalysed ganglioside GM2 degradation

by WT and variant GM2AP. (A) Substrate hydrolysis measured in a

micellar assay system. (B) GM2 degradation measured in a lipo-

somal assay system with LUVs containing 50 mol% PtdCho,

20 mol% Chol, 10 mol% GM2 and 20 mol% BMP. Values repre-

sent means of duplicate measurements; deviations observed were

< 5%.

Fig. 3. Interaction of WT and variant GM2AP with immobilized vesi-

cles measured by SPR spectroscopy. Negatively charged LUVs

containing 20 mol% Chol, 50 mol% PtdCho, 10 mol% GM2 and

20 mol% BMP, were immobilized on a Pioneer L1 sensorchip.

GM2AP (2 lM) was injected at a flow rate of 20 lLÆmin)1 in 50 mM

sodium citrate buffer, pH 4.2, for 180 s, followed by the injection

of protein-free buffer. The measurement started with the protein

injection.

A

B

C

Fig. 1. UV circular dichroism (CD) spectra of wild-type (WT) GM2AP

and variant proteins examined in this study. (A) WT GM2AP and

mutant M117V. (B) WT GM2AP and D113 mutants. (C) WT GM2AP

and E123 mutants. Spectra were obtained at 10 �C, at a protein

concentration of 0.5 mgÆmL)1 in 15 mM sodium phosphate, pH 7.0,

100 mM NaCl.



lized membrane structures via the action of bound

GM2AP [12], either by directly extracting lipids or by

destabilizing the bilayers with a concomitant loss of

lipids. The point mutants examined here were found to

interact with immobilized lipid bilayers in the same

way as WT GM2AP.

To study directly the lipid-transfer properties of

GM2AP, we recently introduced a novel FRET-based

assay system [26]. A new class of fluorescent glyco-

sphingolipid analogue, either 2-(N-(7-nitrobenz-2-oxa-

1,3-diazol-4-yl)) (NBD)–GM1 or 2-NBD–GM2, serves

as FRET-donor for rhodamine–dioleoyl glycero phos-

phoryl ethanolamine (PE) as acceptor which is initially

localized within the same model membrane and thereby

quenches NBD fluorescence. It has been shown previ-

ously that GM1 and GM2 are transferred almost

equally well between vesicles by GM2AP [10]. These

novel fluorescent gangliosides are easily incorporated

into model membranes, where they exhibit an extremely

low ‘off rate’ and are recognized and degraded by HexA

in the presence of GM2AP, similar to their natural ana-

logue. Upon addition of catalytic amounts of GM2AP

to donor vesicles containing NBD–gangliosides, the

activator-mediated glycolipid transfer from donor to

acceptor liposomes increases the NBD fluorescence,

permitting continuous real-time monitoring of GM2AP

transfer activity. As shown in Fig. 4, the point mutants

examined here showed the same ability as WT GM2AP

to transfer NBD–GM1 from donor to acceptor vesicles.

The initial rate of NBD transfer and the capacity

observed under equilibrium conditions are identical.

It has previously been shown that, in the absence of

ganglioside GM2, the GM2 activator protein inhibits

hydrolysis of the synthetic fluorogenic substrate

MUGS by HexA [5]. This negatively charged substrate

can only be degraded by the a subunit of the enzyme,

and the inhibitory effect of GM2AP has been inter-

preted as being indicative of a physical association

between GM2AP and the enzyme which blocks this

subunit’s active site. As shown in Fig. 5, all variant

forms of GM2AP examined show concentration-

dependent inhibition of MUGS degradation. However,

their inhibitory effect is less pronounced than that of

WT GM2AP. The presence of GM2AP and of all

point mutants has no effect on the degradation of the

neutral, unsulfated substrate MUG (data not shown).

Apparently, all GM2AP variants studied here retain

the ability to interact specifically with the a subunit ofthe enzyme. However, the strength of their interaction

with HexA is weaker than for the WT protein, suggest-

ing that the introduced mutations did indeed affect

the binding interface to the enzyme.

Discussion

The GM2 activator protein is a small glycoprotein

cofactor essential for the lysosomal degradation of

ganglioside GM2 by HexA [3]. It mediates the interac-

tion between the water-soluble enzyme and the mem-

brane-embedded glycolipid substrate. Our goal was to

map the protein region on GM2AP responsible for

binding to the enzyme and to further elucidate the

activator’s mode of action at the lipid–water interface.

The capacity of the variant proteins examined to sti-

mulate HexA-catalysed GM2 degradation was meas-

ured in a micellar and a liposomal assay system. In

order to separate the influence of the mutations on the

Fig. 4. Increase in NBD fluorescence through GM2AP-mediated

NBD–GM1 transfer measured in a FRET-based liposomal assay sys-

tem as a function of time. The assay volume of 400 lL contained

8 nmol donor liposomes (20 mol% Chol, 56 mol% PC, 20 mol%

phosphatidic acid, 2 mol% NBD-GM1, 2 mol% rhodamine-PE) and

40 nmol acceptor liposomes (20 mol% Chol, 60 mol% PtdCho,

20 mol% phosphatidic acid) and 37.5 nM WT or variant GM2AP.

Values are means of duplicate measurements, deviations observed

were always < 5%.

Fig. 5. Competition experiment. Degradation of the synthetic fluo-

rogenic substrate MUGS by HexA is inhibited in the presence of

GM2AP in a concentration-dependent manner, suggesting that

HexA and GM2AP form a complex which sterically blocks the act-

ive site of the HexA a subunit. In the presence of GM2AP variants,

MUGS degradation is reduced to a lesser extent, indicating that the

physical association of HexA with these mutant forms is weaker.



lipid- and enzyme-binding properties, we tested the

membrane activity of GM2AP using SPR spectroscopy

and probed the lipid-transfer properties in a FRET-

based assay system. The ability to bind specifically the

a subunit of HexA could be assessed by studying

the competition of GM2AP and its variants with the

hydrolysis of the synthetic fluorogenic hexosaminidase

substrate MUGS.

Our data clearly indicate that mutations introduced

into the a-helical region of GM2AP drastically

decreased the ability of the activator to stimulate

HexA-catalysed degradation of GM2. However, the

membrane activity of these variants, as well as their

lipid-binding and transfer ability, was identical to that

of the WT protein. All variants could still bind speci-

fically to the a subunit of HexA, but their association

with the enzyme was weaker than for WT GM2AP.

This clearly identifies the region affected by these

mutations, the single a helix of GM2AP, as the major

surface epitope interacting with the enzyme.

That the mutants D113K and E123K exhibit the

most drastically reduced stimulatory activity for GM2

degradation and show the weakest binding to HexA

might be attributed to the reversal of local charge

resulting from the exchange of acidic amino acids to

lysine. In addition to possible electrostatic repulsion,

all salt bridges and hydrogen bonds which are possible

between positively charged residues on HexA and

amino acids D113 and E123 in WT GM2AP can no

longer be formed.

Figure 6 shows the position of the examined point

mutations at the interface between activator and HexA,

as a result of theoretical docking studies based on the

known crystal structures of GM2AP [20–22] and HexB

[24,25]. Our results are consistent with the location of

the ceramide tail of GM2 as identified by crystallo-

graphic analysis of lipid complexes of GM2AP [21],

which positions the tetrasaccharide head-group of the

substrate in direct vicinity of the enzyme’s active site.

Among patients with variant AB of GM2 gangliosi-

dosis, whose severe neurological storage of ganglioside

GM2 results from the functional deficiency of

GM2AP, no case has been found in which the muta-

tion occurred directly within the a-helical region of

GM2AP. However, the biochemical consequence of

one fatal mutation, the amino acid exchange C138R,

was thought to be mainly a disrupted interaction with

the enzyme [17], and this might well be affected by

altered orientation of the helix. The crystal structure of

GM2AP [20,21] and the known disulfide bond connec-

tivity [27] indicate that the disulfide bond formed by

C138 with C112 forms a ‘clamp’ that structurally

restrains the activator’s helical domain. It is therefore

most likely that the mutation C138R distorts the orien-

tation of the helix and compromises the activator’s

ability to interact with the enzyme.

In addition to mapping the activator’s binding inter-

face with the enzyme, our data confirm the critical

importance of distinct protein–protein interactions in

lysosomal GM2 degradation. The activator’s role is

not limited to that of a biological detergent that simply

disrupts the membrane structure. In contrast, the acti-

vator ⁄ glycolipid complex represents the true Michaelis

substrate for the degrading enzyme.

Experimental procedures

Materials

For insect cell culture, serum-free IPL-41 medium was pur-

chased from Invitrogen Life Technologies (Carlsbad, CA).

The baculovirus transfer vector pAcMP3, linearized baculo-

virus DNA (BaculoGold), the transfection kit and the insect

cell lines were from Pharmingen, obtained via BD Bio-

sciences (Erembodegem, Belgium). Phosphatidylcholine (egg

yolk, PtdCho) and cholesterol (Chol) were purchased from

Sigma (Taufkirchen, Germany). BMP was obtained from

Avanti Polar Lipids (Alabaster, AL), Lichroprep RP18 was

Fig. 6. Model for the interface in the ternary complex of GM2AP,

GM2 and HexA resulting from theoretical docking studies based on

the known crystal structures of GM2AP [20–22] and HexB [25]. In

the GM2AP structure b sheets are blue, the single a helix is green,

and regions exhibiting conformational differences in free and ligand-

bound form are red. The position of the bound ligand GM2 follows

the model presented in Fig. 4 of Wright et al. [21], as far as the

lipid tail is concerned. Regarding the tetrasaccharide head-group,

however, we consider a more ‘strained’ orientation as presented

here, more likely. The picture was generated using MOLSCRIPT [30].



from Merck (Darmstadt, Germany). The Pioneer L1 Chip

was purchased from Biacore (Uppsala, Sweden). All other

chemicals and solvents were of analytical grade or the high-

est purity available.

Protein expression and mutagenesis

Recombinant GM2AP was expressed in BEVS as described

previously [28]. Site-directed mutagenesis using the vector

pAcMP3-GM2APHis6 [28] as a template was performed

using the QuikChange site-directed mutagenesis kit from

Stratagene (La Jolla, CA). Synthetic oligonucleotides were

obtained from Sigma and Invitrogen Life Technologies.

Mutations encoding the following amino acid exchanges

were introduced into the cDNA of GM2AP: D113K,

D113A, D113Y, M117V, E123K, E123A, E123Y. All

expression vectors encoded a hexahistidine tag at the C-ter-

minus of GM2AP. The resulting constructs encoding point

mutants of GM2AP were fully sequenced using an ABI 310

sequencer and the BigDye cycle sequencing kit, both from

Applied Biosystems (Foster City, CA). Recombinant bacu-

loviruses were then generated by cotransfection of Sf9 cells

with the respective transfer vector and linearized BaculoGold

viral DNA according to the manufacturer’s protocol. Pure

viral stocks were obtained using the end-point dilution

method.

For protein expression, Sf9 cells grown in serum-free

IPL-41 to a cell density of 1.5 · 106 cellsÆmL)1 were infec-

ted with a viral stock at a multiplicity of infection of 5. The

medium was harvested 96 h after infection.

Protein purification

Proteins were purified, with modification, following the

method described by Wendeler et al. [28]. Briefly, the super-

natant was first subjected to perfusion chromatography on

a cation exchange resin (Poros HS) using a BIOCADSprint

System HPLC workstation (Applied Biosystems). Fractions

containing recombinant GM2AP were combined and then

further purified by immobilized metal ion affinity chroma-

tography on Ni-NTA-agarose (Qiagen, Hilden, Germany).

The eluted proteins were analysed by electrophoresis on a

12.5% tricine-SDS ⁄ polyacrylamide gel and visualized by

silver staining. The polyclonal antibody raised against WT

GM2AP [29] recognized all GM2AP mutants. Amino acid

exchanges in the recombinant proteins were confirmed by

ESI-TOF-MS.

Micellar GM2AP assay

In a micellar in vitro system, the activity of recombinant

GM2AP was tested by measuring the stimulation of hex-

osaminidase A-catalysed hydrolysis of [3H]GM2 tritium-

labelled in its terminal GalNAc moiety [3]. In 40 lL of

100 mm sodium citrate buffer containing 2.5 lg BSA,

10 nmol [3H]GM2 were incubated with 80 mU HexA (puri-

fied from post mortem human liver) in the presence of 3 lgrecombinant GM2AP for 1 h. Reactions were stopped by

adding 40 lL methanol. Liberated [3H]GalNAc was isola-

ted using self-packed RP18 cartridges: 0.5 mL LiChroprep

RP18 (Merck) was applied to a glass Pasteur pipette, pre-

viously stuffed with a small amount of glass wool. The

material was equilibrated by washing subsequently with

2 · 1 mL chloroform ⁄methanol 1 : 1 (v ⁄ v), 2 · 1 mL

methanol and 2 · 1 mL chlorofom ⁄methanol ⁄ 0.1 m KCl

3 : 47 : 48 (v ⁄ v ⁄ v). The assay solution was then applied

and the flow-through collected. Soluble [3H]GalNAc was

then eluted with 2 · 1 mL water, and the eluate combined

with the flow-through and 10 mL scintillation liquid.

Radioactivity in the effluents was measured in a scintilla-

tion counter (Packard). One activator unit is defined as the

amount of GM2AP that stimulates the degradation of

1 nmol GM2 per minute and enzyme unit.

Liposomal assay systems

To mimic more closely the reaction conditions encountered

on intralysosomal vesicles and membrane structures, the

degradation of membrane-bound ganglioside GM2 by hex-

osaminidase and GM2AP was studied in a detergent-free,

liposomal assay system as described previously [12].

Vesicle prepapration

Large unilamellar vesicles (LUVs) of 100 nm were prepared

by the following procedure. Appropriate aliquots of the

lipid solutions PtdCho (50 mm, toluol ⁄ ethanol 2 : 1 v ⁄ v),BMP (10 mm, chloroform ⁄methanol 1 : 1 v ⁄ v), Chol

(25.6 mm, chloroform ⁄methanol 2 : 1 v ⁄ v) and GM2 trit-

ium-labelled in its terminal GalNAc moiety (0.5 mm, tolu-

ol ⁄ ethanol 1 : 1 v ⁄ v) were mixed and dried in a stream of

nitrogen. The lipid mixture was dissolved to a total lipid

concentration of 2 mm in sodium citrate buffer (50 mm,

pH 4.2) and freeze–thawed 10 times in liquid nitrogen to

ensure solute equilibration between trapped and bulk

solutions. Unilamellar vesicles were prepared by passage

through two polycarbonate filters (pore size, 100 nm; Aves-

tin) mounted in tandem in a mini-extruder (Liposo-Fast;

Avestin, Ottawa, Canada) a total of 19 times.

Liposomal assay

The standard incubation mixture using GM2 as substrate

contained the following components in a final volume of

50 lL: BSA (50 lgÆmL)1), sodium citrate buffer (pH 4.2,

50 mm), unilamellar liposomes (total lipid concentration:

1 mm), GM2AP (2 lm) and HexA (25 mU). Liposomes

had the following composition: [3H]GM2 (10 mol%,



1.8 CiÆmol)1), Chol (20 mol%), PtdCho (50 mol%) and

BMP (20 mol%). The standard incubation conditions were

37 �C for 30 min, and the enzyme assays were stopped by

the addition of 50 lL methanol. Terminated enzyme assays

were loaded onto a reverse-phase column (RP18, 1 mL)

equilibrated with a solution of chloroform ⁄methanol ⁄ 0.1 m

KCl (3 : 48 : 47, v ⁄ v ⁄ v). The column was eluted with 2 mL

of the same solvent, and the radioactivity in the effluents

was measured in a scintillation counter.

Determination of HexA activity with the

fluorogenic substrates 4-methylumbelliferyl-

2-acetamido-2-deoxy-b-D-glucopyranoside and its

sulfated derivative MUGS

The activity of HexA towards the synthetic substrates

4-methylumbelliferyl-2-acetamido-2-deoxy-b-d-glucopyrano-side (MUG) and its sulfated derivative MUGS was meas-

ured essentially as described previously [5]. For routine

activity measurements, MUG was used as substrate under

the following standard conditions. In a volume of 40 lL,10 mm citrate buffer, pH 4.2, 2 mm substrate, 6 lg BSA

and an appropriate amount of HexA were incubated at

37 �C for 30 min. One enzyme unit is defined as the

amount of hexosaminidase that generates 1 lmolÆmin)1 of

4-methylumbelliferone.

MUGS was used in competition experiments designed to

study the association of GM2AP with the a subunit of

HexA. In a volume of 40 lL, 10 mm citrate buffer, pH 4.2,

2 · 10)5 m substrate, 6 lg BSA and an appropriate amount

of HexA were incubated at 37 �C for 30 min in the absence

or presence of various amounts of GM2AP (up to 10 lg).Reactions were stopped by the addition of 5 vol. of a 0.2 m

glycine, 0.2 m Na2CO3 solution and the generated 4-methyl-

umbelliferone determined by measuring the fluorescence at

440 nm after excitation at 365 nm.

UV CD spectroscopy

CD spectroscopy was used to monitor potential distur-

bances of the protein fold upon introduction of the individ-

ual mutations. CD spectra in the range of 250 to 200 nm

were acquired on a JASCO J600 spectropolarimeter with

four accumulations in a 0.1 cm cuvette at 10 �C for solu-

tions of 0.5 mgÆmL)1 of the mutant proteins in 15 mm

sodium phosphate, 100 mm NaCl, pH 7. Data processing

and evaluation was carried out with jasco software as

recommended by the manufacturer.

SPR spectroscopy

Biomolecular interaction analyses (BIA, SPR spectroscopy)

were carried out at 25 �C with a Bialite instrument (Bia-

core) on a Pioneer L1 Chip. For SPR spectroscopy, lipo-

somes were prepared as described above, but diluted to a

lipid concentration of 0.5 mm in NaCl ⁄Pi (10 mm phos-

phate, 140 mm NaCl, 10 mm KCl pH 7.4). These liposomes

contained unlabelled GM2.

First, LUVs (with a total lipid concentration of 0.5 mm),

diluted in NaCl ⁄Pi, were immobilized on the sensorchip by

two injections at a flow rate of 5 lLÆmin)1 (first injection

60 lL, second injection 20 lL) as described previously [12].

This resulted in a RU shift of 5000–7000 RU. Ten micro-

litres of NaOH (25 mm) was then injected at 100 lLÆmin)1

to remove multilamellar structures and to stabilize the base-

line, resulting in a RU shift of ~ 20–50 RU. Sixty micro-

litres of GM2AP (1 or 2 lm) in running buffer (50 mm

sodium citrate buffer, pH 4.2) were injected into the flow

cells at a rate of 20 lLÆmin)1 with a dissociation time of

180 s. Measurements started with the injection of GM2AP.

FRET-based assay for GM2AP

The fluorescent analogue 2-NBD–GM1 was synthesized as

described previously [26]. Large unilamellar donor vesicles

were prepared with the following composition: PtdCho

(56 mol%), Chol (20 mol%), phosphatidic acid (20 mol%),

2-NBD–GM1 (2 mol%) and rhodamine–PE (2 mol%).

Acceptor vesicles comprised PtdCho (60 mol%), Chol

(20 mol%) and phosphatidic acid (20 mol%). The final

donor vesicle concentration in the assay mixture was

20 nmolÆmL)1, the concentration of acceptor vesicles was

100 nmolÆmL)1 in a total volume of 400 lL in 50 mm citrate

buffer, pH 4.2. The transfer of NBD–GM1 was started by

the addition of GM2AP to a final concentration of 37.5 nm

(WT or point mutants). Fluorescence measurements were

performed at 28 �C using a quartz cuvette in a Shimadzu

RF5000 instrument (Kyoto, Japan) with an excitation wave-

length of 480 nm and an emission wavelength of 522 nm.

Without the addition of GM2AP, no increase in fluorescence

was observed over a period of 2 h. For each experimental

time point over the interval ranging from 0.5 to 30 min, the

instrument’s shutter was opened for only 3–4 s, resulting in

a total of maximal 52 s illumination of NBD–GM2. Under

these conditions, photobleaching was negligible.

Acknowledgements

We thank Dr Christina Schuette, Luebeck, for provi-

ding purified HexA, and Dr Joerg Hoernschemeyer,

Loerrach, for performing ESI-TOF-MS. This study

was supported by the Deutsche Forschungsgemeinsc-

haft, SFB 284 and SA-257 ⁄21-1.

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The enzyme-binding region of human GM2-activator protein

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