Computational analysis of human N-acetylgalactosamine-6-sulfatesulfatase enzyme: an update in genotype–phenotype correlationfor Morquio A

Sergio Olarte-Avellaneda • Alexander Rodrıguez-Lopez •

Carlos Javier Almeciga-Dıaz • Luis Alejandro Barrera

Received: 3 March 2014 / Accepted: 21 April 2014 / Published online: 7 October 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Mucopolysaccharidosis IV A (MPS IV A) is a

lysosomal storage disease produced by the deficiency of N-

acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme.

Although genotype–phenotype correlations have been

reported, these approaches have not enabled to establish a

complete genotype–phenotype correlation, and they have

not considered a ligand–enzyme interaction. In this study,

we expanded the in silico evaluation of GALNS mutations

by using several bioinformatics tools. Tertiary GALNS

structure was modeled and used for molecular docking

against galactose-6-sulfate, N-acetylgalactosamine-6-sul-

fate, keratan sulfate, chondroitin-6-sulfate, and the artificial

substrate 4-methylumbelliferyl-b-D-galactopyranoside-6-

sulfate. Furthermore, we considered the evolutionary resi-

due conservation, change conservativeness, position within

GALNS structure, and the impact of amino acid substitu-

tion on the structure and function of GALNS. Molecular

docking showed that amino acids involved in ligand

interaction correlated with those observed in other human

sulfatases, and mutations within the active cavity reduced

affinity of all evaluated ligands. Combination of several

bioinformatics approaches allowed to explaine 90 % of the

missense mutations affecting GALNS, and the prediction

of the phenotype for another 21 missense mutations. In

summary, we have shown for the first time a docking

evaluation of natural and artificial ligands for human

GALNS, and proposed an update in genotype–phenotype

correlation for Morquio A, based on the use of multiple

parameters to predict the disease severity.

Keywords MPS IV A � GALNS � Keratan sulfate �Chondroitin-6-sulfate � Molecular modeling �Computational molecular docking

Introduction

N-Acetylgalactosamine-6-sulfate sulfatase (GALNS, EC

3.1.6.4, UniProt P34059) is a lysosomal enzyme involved

in the degradation of glycosaminoglycans (GAG) keratan

sulfate (KS) and chondroitin-6-sulfate (C6S) [41]. Muta-

tions in GALNS gene lead to Mucopolysaccharidosis IV A

(MPS IV A, Morquio A disease, OMIM 253000), in which

both KS and C6S are accumulated within the lysosome

[41]. MPS IV A is a lysosomal storage disease (LSD) with

an estimated incidence of 1:200.000, and characterized by

systemic skeletal dysplasia, laxity of joints, hearing loss,

corneal clouding, valvular heart disease, and pulmonary

dysfunction [20, 41]. Currently, there are not corrective

therapies for MPS IV A patients, and only supportive

measures and surgical interventions are used to treat some

disease manifestations [20, 41]. However, enzyme

replacement therapy [6, 9, 21, 27, 39, 40] and gene therapy

are under evaluation [2, 3, 14].

The full-length GALNS cDNA has been cloned and

encodes a 522 amino acids protein including a signal

S. Olarte-Avellaneda

Clinical Bacteriology Program, School of Health Sciences,

Universidad Colegio Mayor de Cundinamarca, Bogota, D.C.,

Colombia

S. Olarte-Avellaneda � A. Rodrıguez-Lopez (&) �C. J. Almeciga-Dıaz (&) � L. A. Barrera

Proteins Expression and Purification Laboratory, Institute for the

Study of Inborn Errors of Metabolism, School of Sciences,

Pontificia Universidad Javeriana, Kra 7 No. 43-82 Building 53,

Room 303A, Bogota, D.C., Colombia

e-mail: rodriguez.edwin@javeriana.edu.co

C. J. Almeciga-Dıaz

e-mail: cjalmeciga@javeriana.edu.co

123

Mol Biol Rep (2014) 41:7073–7088

DOI 10.1007/s11033-014-3383-3

peptide of 26 residues [36]. Over 170 mutations have been

described in GALNS gene, including missense/nonsense

mutations (75 %), splicing mutations (8 %), small dele-

tions (10 %), small insertions (1.7 %), small indel (\1 %),

gross deletions (1.7 %), gross insertions/duplications

(1 %), and complex rearrangements (1 %) [32]. GALNS is

a homodimer of 120 kDa, with monomers of 60 kDa

formed by 40 and 15 kDa polypeptides bound by a disul-

fide bond [18], containing two N-glysosylations [26]. As

other lysosomal enzymes, GALNS has a signal peptide that

leads the nascent protein to the endoplasmic reticulum and

Golgi apparatus system, in which the protein is modified

through N-glycosylation and proteolytic processing [15].

The most important posttranslational GALNS modifica-

tion, common to other sulfatases, is the active-site activa-

tion through the conversion of cysteine 79 to formylglycine

(FGly), in a process mediated by the formylglycine-gen-

erating enzyme [5, 38]. The tertiary structure model of

GALNS showed that it has two domains: a large N-ter-

minal domain with a a/b topology with a 10-stranded b-

sheet and six a-helices, and a small C-terminal domain

comprising a 4-stranded antiparallel b-sheet perpendicular

to a long a-helix [26, 34]. The catalytic residue (FGly) lies

at the bottom of a cavity containing a metal ion (Ca2?) and

is flanked by positively charged amino acids, as observed

in other sulfatases [12, 26, 34].

Despite the high heterogeneity of Morquio A patients,

some genotype–phenotype correlations have been estab-

lished. So far, four mutations groups have been identified:

(i) mutations causing destruction of the hydrophobic core

or modification of the packing, (ii) removal of a salt bridge

to destabilize the entire conformation, (iii) modification of

the active site geometry, and (iv) location on surface of the

protein to alter lysosomal targeting, hydrogen bounds, or

N-glycosylation sites [34]. Mutations affecting the hydro-

phobic core, salt bridges, or active cavity (activation or

folding) were associated with severe phenotypes; while

mutations located on protein surface or distant from the

active cavity, as well as those affecting the N-glycosyla-

tions, were associated with attenuated phenotypes [19, 34,

38]. A phenotype–genotype correlation approach, based on

the evolutionary conservation of amino acids, conserva-

tiveness of amino acids changes (according to amino acids

properties), and structural conservation of amino acid res-

idues, was also reported [37]. The results showed that

missense mutations are mainly located in highly conserved

residues and can lead to severe phenotypes if they are the

result of a non-conservative amino acid exchange. A

structural and functional analysis of missense mutations in

GALNS enzyme by using several bioinformatics tools

showed that mutations could produce structural alterations,

changes in ligand affinity (N-acetyl-galactosamine-6-sul-

fate), and the presence of a positive correlation between

mutations in hydrophobic cores and severe phenotype [33].

Finally, mapping of missense mutations in a X-ray crystal

structure revealed that most of the mutations affect the

hydrophobic core of the structure [26]. Recently, correla-

tions between genotype, phenotype, and blood and urine

KS levels were proposed, which allowed to predict the

clinical severity more precisely [8].

To increase the knowledge in the genotype–phenotype

correlation for MPS IV A and to understand the GALNS–

ligand interactions, in this study, we used several bioin-

formatics tools to expand the in silico evaluation of

GALNS and the effect of mutations on structure and pro-

tein–ligand interactions. A docking study with natural and

artificial ligands was carried out for the wildtype and

mutated GALNS models. The results suggest the interac-

tion of the evaluated ligands with the residues Asp39,

Asp40, FGly79, Arg83, Tyr108, His142, His236, Asp288,

Asn289, and Lys310. Combination of several bioinfor-

matics approaches allowed explaining 90 % of missense

mutations, and the prediction of the phenotype for 21

missense mutations.

Methods

Protein structure modeling

GALNS protein was retrieved from Uniprot (Entry No.

P34059), and signal peptide and potential N-glycosylation

sites were predicted using SignalP 4.0 and NetNGlyc 1.0,

respectively, available at Expasy server. GALNS tertiary

structure without signal peptide was modeled by using

I-TASSER [44], selecting the tertiary structure of human

arylsulfatase A (ASA, PDB 1AUK) as a template for protein

threading. Protein structure was validated using PDBsum

[17]. Calcium ion was added to the tertiary structure using

YASARA View v11.4.18 (YASARA Biosciences GmbH,

Vienna, Austria), constrained to Asp39, Asp40, Asp288, and

Asn289, as previously reported for ASA [30]. Amino acids

interacting with Ca?2 were predicted using Protein Structure

Analysis Package (PSAP) [4]. Active site amino acids and

volume of cavity were predicted using Computed Atlas of

Surface Topography of Proteins (CASTp) [7]. Structures

were visualized with YASARA View v11.4.18 and UCSF

Chimera v1.6.2 [23], while Swiss-PdbViewer v4.1 [13] was

used for energy minimization and to calculate the root-mean-

square deviation (RMSD).

MPS IV A mutations and phenotypes

Missense mutations affecting GALNS gene were retrieved

from Human Gene Mutation Database (HGMD) [32], as

well as from previous reports [16, 26, 37, 38], where

7074 Mol Biol Rep (2014) 41:7073–7088

123

phenotype was defined for each mutation. We did not

consider deletions and nonsense and splice-site mutations,

since they have been previously associated with severe

phenotypes due to the synthesis of a truncated enzyme or

changes in the open reading frame [38].

Molecular docking

Molecular docking of the modeled GALNS structure and

the natural and artificial ligands was done using Molegro

Virtual Docker v5.5 (MVD, CLC bio, Aarhus N, Den-

mark). The search space was centered between Cys79 and

calcium ion, and radius of 7, 9, 12 and 15 A were evalu-

ated. Structures of galactose-6-sulfate (G6S) and keratan

sulfate (KS, 1KES) were retrieved from the RCSB-Protein

Data Bank, while structures of N-acetylgalactosamine-6-

sulfate (6S-GalNAc, CID 193456) and chondroitin-6-sul-

fate (C6S, CID 24766) were retrieved from PubChem.

Structure of the artificial ligand 4-methylumbelliferyl-b-D-

galactopyranoside-6-sulfate (4MUGPS), which is used for

GALNS enzyme activity assay [42], was built using Mar-

vinSketch at Marvin Suite (ChemAxon Ltd., Budapest,

Hungary). Docking for each ligand was run 20 times and

constrained to the active cavity. To evaluate the effect of

mutations on GALNS-ligand affinity fifteen mutations

affecting amino acids present within the active cavity were

studied. Mutations were manually included into GALNS

model by using Swiss-PdbViewer v4.1, and interactions of

mutated models with ligands were evaluated as described

above for the natural and artificial substrates.

Molecular dynamics

Molecular dynamics analysis were done for the docking

results obtained for wild-type GALNS against G6S and 6S-

GalNAc. Simulations were developed using GROMACS

4.5.5 [24], and topologies of ligands were generated using

PRODRG2 [31]. Simulations were carried out for 20 ns,

and trajectories were analyzed by RMSD and root mean

square fluctuation (RMSF) of the ligand. All simulations

were done at the High Performance Computing Center—

ZINE—of Pontificia Universidad Javeriana.

Genotype–phenotype correlation

To evaluate the genotype–phenotype correlation of muta-

tions outside the active cavity, the sequence of the human

GALNS enzyme was separately aligned with GALNS

sequences from other species (Macaca mulata AFE69926.1,

Bos taurus NP_001193258.1, Rattus norvergicus NP_00104

1316.1, Canis lupus familiaris NP_001041585.1, Mus mus-

culus AAH04002.1, Mustela putorius furo AER98880.1,

Cricetulus griseus EGV94073.1, Heterocephalus glaber

EHB15848.1, Gallus gallus XP_414208.1, Loxodonta afri-

cana XP_003418184.1, Meleagris gallopavo XP_003209

945.1, Xenopus (Silurana) tropicalis XP_002933701.1,

Saccoglossus kowalevskii XP_002736267.1, Oreochromis

niloticus XP_003445750.1, Amphimedon queenslandica

XP_003383435.1, Sus scrofa NP_999120.1, Cavia porcellus

XP_003460959.1, Equus caballus XP_001488119.1, Anolis

carolinensis XP_003229531.1), or 17 human sulfatases

(available at http://sulfabase.tigem.it/Hs.html). Sequences

were aligned using Muscle [10], and the phylogenetic tree

was generated by using MEGA5 [35]. For genotype–phe-

notype correlation it was considered evolutionary residue

conservation in GALNS and sulfatases sequences, residue

change conservativeness, residue position within GALNS

structure, Polyphen2 results [1], and values of Atomic

Accessible Surface Area (AASA) as previously reported

[26]. Swiss-PdbViewer v4.1 was used to include the mis-

sense mutations into the GALNS 3D model and for estima-

tion of energy minimization.

Statistical analysis

Differences between groups were tested for statistical sig-

nificance by using Student’s t test. An error level of 5 %

(p \ 0.05) was considered significant. Analyses were per-

formed using SPSS Statistics v18 (IBM Corp, Armonk, NY).

Results and discussion

In this study, we carried out a computational evaluation of

human GALNS missense mutations and their genotype–

phenotype correlation in Morquio A disease. Although

several studies have carried out this task [8, 26, 33, 34, 37],

there are some issues that have not been completely evalu-

ated. For example, the study of ligand affinity by molecular

docking of a modeled wildtype and mutated GALNS did not

consider the presence of calcium ion within the active cavity

[33]. Furthermore, the three dimensional structure of human

GALNS, determined by X-ray crystallography, was only

used for localization of the mutations, and the ligand binding

analysis with N-acetylgalactosamine was not possible since

the sugar was bound in a nonproductive orientation [26].

Thus, it is still necessary to expand the in silico evaluation of

GALNS, specially the ligand–enzyme interaction, in order to

increase the knowledge in the genotype–phenotype corre-

lation for Morquio A.

3D model analysis

The sequence of the signal peptide (26 amino acids) was

removed from GALNS sequence, and the remained amino

acids (522) were used to predict the 3D structure based on

Mol Biol Rep (2014) 41:7073–7088 7075

123

the reported structure of ASA. Although GALNS and ASA

only have a 34 % identity, the phylogenetic study (see

above results) showed that ASA is the closest human sul-

fatase to GALNS. The final model had a C-score of 0.38

and presented the 522 amino acids of the primary structure,

conforming 8 a-helices and 23 b-sheets (Fig. 1a), which

are similar to the results of crystalized GALNS (9 a-helices

and 23 b-sheets) [26]. Ramachandram plot analysis of the

model showed that 97.9 % of amino acids were in the two

most favored regions, which is similar to the results

obtained for ASA (98.7 %) and previously modeled human

GALNS (96.8 %) [33]. G-factors analysis showed an

average score of -0.34, suggesting that dihedral angles and

main-chain covalent forces are not unusual (Values below

-0.5: unusual, and below -1.0: highly unusual).

During the final analysis of this study, the crystal

structure of human GALNS was published [26]. The

modeled human GALNS has a RMSD of 1.2 A (399

atoms) against the crystal structure of human GALNS

(Fig. 1b). The main differences were observed on the

C-terminal end and four loops located at the protein surface

(Fig. 1b), as previously reported for other sulfatases [12].

The modeled GALNS has a RMSD of 0.65 A (451 atoms),

1.24 A (333 atoms) and 1.22 A (391 atoms) against ASA,

Arylsulfatase B (ARSB, PDB 1FSU), and ARSC (PDB

1P49), respectively, while crystallized GALNS has a

RMSD against the same sulfatases of 1.31 A (394 atoms),

1.37 A (315 atoms), and 1.24 (367 atoms) A, respectively.

As it has been observed for all human sulfatases,

GALNS presents the characteristic conversion of Cys to

Formylglycine (FGly) at the active site, mediated by the

formylglycine-generating enzyme [29]. This Cys-to-FGly

modification was not included in the final GALNS 3D

model since docking software used does not recognize the

FGly as an amino acid but it does as a ligand, interfering

with the docking analysis.

The active cavity on model GALNS had an area of

1,152 A2 and a volume of 1,244.6 A3 in which 58 amino

acids were involved. The residues of the primary active site

described for the X-ray GALNS model [26], Asp39,

Asp40, FGly79 (Cys79), Arg83, Tyr108, His142, His236,

Asp288, Asn289 and Lys310 are presented within the

predicted active cavity (Fig. 1c). These residues have been

also identified within the active cavity of ASA, ARSB,

ARSC, and a homologous arylsulfatase from Pseudomonas

aeruginosa [12]. Overall, the spatial arrangement of amino

acids within the active cavity was similar between modeled

and crystallized GALNS (Fig. 1c), showing the high

degree of conservation of the active cavity, as previously

described for other sulfatases [12]. Recently, it was

reported the computational analysis of the active cavity of

GALNS from human and other eight species (i.e. rhesus

macaque, bovine, mouse, rat, dog, chicken, tilapia nilotica,

and pig) [22]. The results showed that 85 % of amino acids

of the active cavity were conserved among the studied

species, and that active cavity of these enzymes has a

Fig. 1 Human GALNS tertiary

structure. a Tertiary structure of

GALNS was modeled using the

tertiary structure of the human

Arylsulfatase A (ASA, PDB

1AUK) as a template for protein

threading. Green sphere

represents calcium ion present

at the bottom of the active

cavity. b Modeled human

GALNS (red) has a RMSD of

1.2 A against the crystal

structure of human GALNS

(blue). Main differences

between both structures are

pointed within gray circules.

Spheres represent calcium ions

present at the cavity. c Amino

acids present at the active cavity

in modeled (red) and crystal

(blue) GALNS. d Active cavity

of modeled human GALNS,

with calcium ion located at the

botton of the cavity. (Color

figure online)

7076 Mol Biol Rep (2014) 41:7073–7088

123

positive partial charge, which correlates with the negative

charge of GALNS substrates [11].

Since presence of calcium ion within the active cavity

has been reported for GALNS [26, 34], and other human

sulfatases [12], this divalent ion was included within

modeled structure, and was located at the bottom of the

active cavity (Fig. 1d). PSAP analysis showed that Ca2?

formed a coordination complex with Asp39, Asp40,

Asp288, and Asp289, which correlates with the amino

acids reported for GALNS [26] and ARSC [12].

In summary, these results show the quality of the pre-

dicted 3D structure of the human GALNS and the high

conservation of the tertiary structure of sulfatases [12, 26],

and that the predicted GALNS model could be used to

study the effect of missense mutations on enzyme function

and structure.

Docking analysis

Enzyme–ligand interaction was evaluated by using struc-

tures of the natural ligands KS and C6S, monomers G6S

and 6S-GalNAc, and the artificial ligand 4MUGPS. For the

evaluated ligands, it was expected that sulfate would be

located at the bottom of the cavity close to the active

residue [30]. Among natural ligands, only C6S was able to

fit within the active cavity, interacting with Ca2?, Leu78,

Cys79, Thr100, Ala102, His103, His142, Tyr181, Lys310,

Asn407, and Trp409, through H-bonds and electrostatic

and steric interactions (Fig. 2; Table 1), with an affinity

energy of -173.337 kJ/mol. Docking of monomers (G6S

and 6S-GalNAc), and the artificial ligand are shown in

Fig. 3 and summarized in Table 1. Monomers showed

lower affinity energy than that of the artificial ligand, while

4MUGPS showed lower affinity for the enzyme than that of

C6S, suggested by a higher energy of affinity of 4MUGPS

than that of C6S (-121.685 vs. -173.337 kJ/mol), as

described by natural and artificial ligands of ASA [30]. For

the evaluated ligands, the sulfate group interacted with

Ca?2, Cys79, Arg83, His142, and Asp288/Asn289, while

Ala102 and Tyr108 or Tyr181 interacted with the carbon

chain, suggesting the importance of these residues in

enzyme–ligand interaction. It was proposed for ARSC that

Arg79, Lys134, His136, His290, and Lys368, which cor-

respond to Arg83, Lys140, His142, His236, and Lys310

within GALNS structure, participate in the catalytic

mechanism of ARSC, and could be extended to the other

sulfatases [12]. In fact, similar residues have been involved

in ligand binding of ASA [30]. Analysis G6S and 6S-

GalNAc interaction with GALNS from rhesus macaque,

bovine, mouse, rat, dog, chicken, tilapia nilotica, and pig,

showed a lower affinity than that observed for human

GALNS [22]. Futhermore, amino acids involved in inter-

action with the sulfate group of G6S and 6S-GalNAc were

highly conserved among studied structures, although in

non-human GALNS new residues interacted with carbon

chain of the ligands [22].

Molecular dynamics analysis of G6S and 6S-GalNAc

showed that ligands RMSD were 1.05 ± 0.17 A and

0.91 ± 0.16 A, respectively (Fig. 4a), suggesting that

ligands position was stable during the evaluated time

(20 ns). Furthermore, RMSF showed that deviation of

atoms for each ligand along the evaluated time was

0.63 ± 0.39 and 0.45 ± 0.27 A for G6S and 6S-GalNAc

(Fig. 4b, c), respectively. Speficically, for G6S the atoms

with the highest deviation (between 1 and 1.2 A) were

oxygens of sulfate group (O2, O3, and O4) as well as

hydrogens H6, H7, and H8 (Fig. 4b). On the other hand, for

6S-GalNAc the highest deviation (*1 A) was observed for

hydrogens H3, H5, and H6 (Fig. 4c). In summary, these

results support the docking results and suggest that this

Fig. 2 Molecular docking for chondroitin-6-sulfate. a 3D represen-

tation of residues interacting with chondroitin-6-sulfate. b 2D repre-

sentation of residues interacting with chondroitin-6-sulfate. Blue lines

hydrogen bonds; green lines electrostatic interactions; red letters

steric interactions. Green sphere represents calcium ion present at the

bottom of the cavity. (Color figure online)

Mol Biol Rep (2014) 41:7073–7088 7077

123

strategy can be used to model the effect of mutations on

ligand–GALNS interactions.

Missense mutations affecting residues located within the

active cavity were modeled, and the resulting mutated

enzymes were analyzed through docking against C6S, G6S,

6S-GalNAc, and 4MUGPS. Results of energy minimiza-

tion, energy of affinity mutations, and intra-molecular

hydrogen bonds for mutations P77R, C79Y, S80L, H142R,

H166Q, G168R, H236D, N289S, G290S, A291T, A291D,

G301C, G309R, K310N, and M318R, are summarized in

Table 2. Six out of 15 mutations affected amino acids

directly involved in the enzyme–ligand interaction

(underlined mutations). In all cases, the mutations pro-

duced an increase in the energy of affinity for the four

evaluated ligands, suggesting a reduction in the GALNS-

ligand affinity. These results differ from those of a previous

Table 1 Summary of the interactions of natural and artificial ligands for human GALNS enzyme. Chondroitin-6-sulfate (C6S), N-acet-

ylgalactosamine-6-sulfate (6S-GalNAc), galactose-6-sulfate (G6S), or 4-Methylumbelliferyl-b-D-galactopyranoside-6-sulfate (4MUGPS)

Ligand Affinity

energy

(kJ/mol)

Interactions

H-bonds Electrostatic Steric

C6S -173.337 Cys79, Thr100, Ala102, His142,

Tyr181, Lys310, and Asn407

Ca2? Leu78, His103, and Trp409

G6S -115.618 Cys79, Arg83, Ala102, Cys165,

Tyr181, Lys310

Ca2?, Asp39, Asp40, His142,

Asp288, Asn289

Ser80, Tyr108

6S-GalNAc -120.104 Cys79, Tyr181, His236, Asn289, Lys310 Ca2?, Asp39, Asp40, Asp288 Leu78, Ala 102, Trp 409

4MUGPS -121.685 Cys79, Thr100, Ala102, Asn289, Lys310 Ca2?, Asp39, Asp40, His236, Asp288 Asn407, Trp409, Glu410, Gln311

Fig. 3 Molecular docking for galactose-6-sulfate (G6S), N-acetylga-

lactosamine-6-sulfate (6S-GalNAc), and 4-Methylumbelliferyl-b-D-

galactopyranoside-6-sulfate (4MUGPS). a, c, d 3D representation of

residues interacting with G6S, 6S-GalNAc and 4MUGPS. b, c, d 2D

resentation of the residues interacting with G6S, 6S-GalNAc and

4MUGPS. Blue lines hydrogen bonds; green lines electrostatic

interactions; red letters steric interactions. Green sphere represents

calcium ion present at the bottom of the cavity. (Color figure online)

7078 Mol Biol Rep (2014) 41:7073–7088

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GALNS docking study, in which a change in the energy of

affinity for some mutations was not observed [33]. The

difference in these results could be associated with the

presence of Ca2? within the present model, which has been

shown to have a positive effect on docking results [30], or

to the software used for the docking analysis (AutoDock

Vina vs. MVD).

A previous report suggested that mutation P77R affects

GALNS through the destruction of a hydrophobic core or a

packing modification [34]. However, we have observed

that this mutation affects GALNS enzyme by alteration of

the ligand–enzyme interaction. Meanwhile for mutations

H166Q and G168R, it has been suggested that they modify

the active cavity [34], which agrees with present results.

Sum of all affinity energies for each mutation showed

that energy levels for attenuated and severe phenotypes

were higher than those obtained for the wild type enzyme

(Table 2; Fig. 5a). Furthermore, the sum of all affinity

energie values for severe mutations trend to be higher than

those obtained for attenuated phenotype mutations.

An important observation is that both K310N and

A291D, two mutations associated with attenuated pheno-

type, produced an increase in the energy of affinity similar

to that observed for the other severe mutations within the

active cavity. Although K310 was identified as a residue

involved in enzyme–ligand interaction, K310N mutation

could be associated with an attenuated phenotype since the

ligand interaction with K310 was not lost in K310N. Ne-

verthelss, a different picture was observed for mutations

A291D (attenuated) and A291T (severe). In wildtype

enzyme, A291 has a hydrogen bond with K310, which was

conserved both in A291D and A291T mutations. However,

different atoms are involved in this hydrogen bond:

between A291 oxygen from backbone and K310 hydrogen

from a-amino in wildtype, and between A291D or A291T

hydrogen from carboxyl group and nitrogen from a-amino

(Fig. 5b), which could affect the orientation of K310.

Furthermore, mutation A291T produced a new hydrogen

bond with G301 that is not observed by mutation A291D

(Fig. 5b). This new hydrogen bond in A291T could affect

further the orientation of K310 leading to a severe phe-

notype, due to the direct role of this residue in the enzyme–

ligand interaction.

Since mutations H236D and N289S affect residues

involved in the ligand–enzyme interaction, it would be

expected that they produce a severe phenotype. However,

these mutations are associated with an attenuated pheno-

type. This phenotype could be explained by the fact that in

both cases the ligand–protein interactions present in the

wild-type enzyme were conserved in the mutated enzymes.

We did not observe a direct correlation between the

energy of minimization, loss/gain of hydrogen bonds (data

not shown), and the phenotype of the disease (Table 2),

suggesting that changes in the affinity energy could be a

better predictor for a phenotype–genotype correlation for

mutations affecting the active cavity.

Fig. 4 Molecular dynamics analysis of wild-type GALNS complexed

with G6S and 6S-GalNAc. Results were evaluated for RMSD of

ligands (a) and root mean square fluctuation (RMSF) of G6S (b) and

6S-GalNAc (c) atoms during 20 ns of molecular dynamics simulation.

Atoms with the highest deviations are shown within each RMSF

figure

Mol Biol Rep (2014) 41:7073–7088 7079

123

Additional computational analysis

Figure 6 shows the phylogenetic trees from the alignment

of GALNS sequences several species and the alignment of

sequences from human sulfatases. One hundred ninety-

eight amino acids were conserved among GALNS

enzymes. As expected, human GALNS was closer to that

of Macaca mulatta and distant from Saccoglossus kowa-

levskii (Acorn worm) and Amphimedon queenslandica

(sponge) (Fig. 6a). On the other hand, 17 amino acids were

conserved among the studied sulfatases, and GALNS was

close related to ASA (Fig. 6b), which support the selection

of this model as template for GALNS modeling. Fifteen out

of the 17 conserved residues for sulfatases were also con-

served among the evaluated GALNS enzymes: C79, P81,

S82, R83, G89, G139, K140, H236, D262, D288, G290,

P327, T348, G365, and D447 (amino acid numbers are

relative to human GALNS sequence), while D39 and G132

were conserved among sulfatases but not in GALNS from

other species.

Taking into account the above results, we analyzed 124

missense mutations reported for GALNS from which 103

have a reported phenotype, 69 and 34 associated to severe

and attenuated phenotypes, respectively (Table 3), while

the remaining 21 mutations lack of a reported phenotype

(Table 4). We considered the amino acid conservation in

human sulfatases and GALNS from different species, res-

idue position within GALNS structure, Polyphen2 results,

energy minimization, and Atomic Accessible Surface Area

(AASA) for the genotype–phenotype correlation.

Sixty-nine out of 103 mutations with assigned pheno-

type (67 %) have a reported severe phenotype. From these

69 mutations, 63 (91 %) were located in highly conserved

residues (present at 15–20 of the studied sequences), while

46 (66 %) were located in completely conserved amino

acids among GALNS sequences. Forty-three out of these

69 mutations (62 %) were produced by a non-conservative

change, with 40 of them located in highly conserved resi-

dues. Finally, 34 out of these 69 mutations were located in

residues highly conserved in both GALNS and sulfatases

sequences.

On the other hand, 34 mutations have an attenuated

reported phenotype (Table 2). Twenty-six out of these 34

(76 %) mutations were located in low conserved amino

acids (present at nine or less sulfatases sequences), 11

(32 %) were produced by a conservative change, and 20

(59 %) were located in the surface of the enzyme.

In summary, these results show that severe mutations

preferably affect higly conserved amino acids and involve

non-conservative amino acid changes; while attenuated

mutations seem to affect less conserved residues, without a

clear preference for conservative or non-conservative

amino acid changes, as previously reported [37].

Energy minimization for missense mutations associated

with severe or attenuated phenotype was obtained after

modeling them within the GALNS 3D-structure (Table 3),

Table 2 Results of energy minimization and affinity energy after

docking of chondroitin-6-sulfate (C6S), N-acetylgalactosamine-6-

sulfate (6S-GalNAc), galactose-6-sulfate (G6S), or

4-Methylumbelliferyl-b-D-galactopyranoside-6-sulfate (4MUGPS)

with wild-type and mutated human GALNS. Parenthesis numbers

represent HGMD accession numbers

Mutation Phenotype Energy

minimization

(kJ/mol)

Affinity energy (kJ/mol)

C6S G6S 6S-GalNAc 4MUGPS Sum

GALNS Normal -12,420 -173.337 -115.618 -120.104 -121.685 -530.744

P77R (CM950524) Severe -12,663 -155.047 -113.530 -117.669 -114.834 -501.080

C79Y (CM045155) Severe -11,994 -161.745 -108.831 -115.234 -109.769 -495.579

S80L (CM970577) Severe -11,370 -159.109 -114.209 -120.016 -110.918 -504.252

H142R Severe -12,624 -159.604 -109.509 -119.295 -99.620 -488.028

H166Q (CM970587) Severe -12,559 -172.787 -113.118 -118.002 -106.561 -510.468

G168R (CM970588) Severe -12,260 -160.336 -113.464 -112.341 -103.364 -489.505

H236D Attenuated -12,369 -149.569 -115.125 -117.141 -107.347 -489.182

N289S Attenuated -12,236 -162.749 -112.261 -114.195 -110.827 -500.032

G290S (CM970594) Severe -12,423 -163.532 -114.135 -119.413 -112.757 -509.837

A291T (CM950535) Severe -12,268 -162.139 -113.600 -114.513 -96.969 -487.221

A291D (CM970595) Attenuated -12,349 -172.340 -113.937 -114.181 -115.690 -516.148

G301C (CM970597) Severe -12,091 -167.525 -115.454 -119.218 -102.139 -504.336

G309R (CM970598) Severe -8,589 -152.197 -103.978 -119.095 -116.071 -491.341

K310N (CM054737) Attenuated -12,569 -167.765 -115.497 -112.741 -119.305 -515.308

M318R (CM950536) Severe -12,667 -163.070 -113.307 -117.472 -103.205 -497.054

7080 Mol Biol Rep (2014) 41:7073–7088

123

excluding those present at the signal peptide (M1V, L15M

and G23R). Seventy-three out of 100 mutations (73 %)

produced an increase in the energy minimization in com-

parison to the wildtype enzyme value, with 48 out of 73

mutations (65 %) associated with a severe phenotype.

Similar results were reported for GALNS [34] and N-

acetylgalactosamine-4-sulfatase [28], showing that a high

percentage of mutations associated with a severe pheno-

type were produced by large structural changes in the

protein structure. However, this value by itself cannot

explain the phenotype associated to the mutation.

We further characterized mutations M41L, D60N, R94C,

R94G, F97V, Q111R, F156S, N204K, H236D, N289S,

K310N, T312S, A324E, and M391V, which are associated

with an attenuated phenotype. Mutations M41L, F97V,

T312S, A324E and M391V were produced by a conservative

change of the amino acid, while D60N, N204K, M391V are

placed in the surface of the protein and are not involved in the

active cavity. In the case of mutation N204K, this affects an

N-glycosylation site which could partially affect the intra-

cellular traffic or the stability of the enzyme [19], since one of

N-glycosylation will remain in the enzyme.

Mutations S80L, S135R, S341R, S470P and T312S are

associated with severe and attenuated phenotypes, and

might be associated with predicted phosphorylation sites.

Mutation S80L (severe phenotype), affects a highly con-

served amino acid among GALNS sequences and is present

within the active cavity of the enzyme, while for S135R

and S341R the severe phenotype could be produced by a

non-conservative change from a polar to a basic amino

acid. Although the T312S (attenuated phenotype [43])

affects a highly conserved amino acid, this residue is not

present within the active cavity. In this case, the mutation

was produced by a conservative change, and the serine

could be potentionally phosphorylated.

Recently, it was reported the missense mutation C165Y,

which was associated with a severe phenotype [16]. The

computational analysis of this mutation confirmed the severe

phenotype. C165Y mutation affects a completely conserved

residue within studied GALNS sequences, C165 is part of the

active cavity interacting through a H-bond with the carbon

chain of the ligand G6S, and the mutant enzyme showed a

value of minimization energy higher (-11,458 kJ/mol) than

that observed for the wildtype enzyme.

As previously reported, combination of studied factors

provides a better association between missense variants

and clinical severity than that obtained by either factor

alone [37]. Sixty-two out of the 69 severe mutations

(89.8 %) could be explained by the combination of residue

conservation among GALNS and sulfatases sequences,

residue change conservativeness, residue position within

GALNS structure, Polyphen2 results, and the value of

minimization energy (Table 2); which is higher than the

obtained by the combination of evolutionary conservation

and conservativeness [37]. The remaining severe mutations

(seven), are located in the C-terminal end that in the crystal

structure showed a novel conformation, where the poly-

peptide reverses direction and forms a wall of the active

cavity contributing to substrate selectivity [26], which

could explain the phenotype associated to these mutations.

To explain the 34 mutations associated with attenuated

phenotype, we added the value of the Atomic Accessible

Surface Area as reported previously [26]. Combining all

studied factors, including the docking results for mutations

N289S, A291D and K310N, allowed explaining 27 out of

the 34 mutations (79.4 %). However, high heterogeneity

was observed for the studied factors, which agrees with

previous reports showing high molecular heterogeneity for

this group of mutations [19].

Finally, we used these bioinformatics tools to predict the

phenotype for 21 mutations without a clear reported phe-

notype (Table 4). Fourteen mutations could be associated

Fig. 5 a Total affinity energy (kJ/mol) for the evaluated ligands and

mutations. Phenotype was assigned according to literature reports

(**p \ 0.001). b Effect of A291D (red) and A291T (yellow) mutations.

Blue residues wild-type enzyme; Blue line hydrogen bonds. (Color

figure online)

Mol Biol Rep (2014) 41:7073–7088 7081

123

with a severe phenotype, since they affect 13 highly to

completely conserved residues and have a minimization

energy value higher than that of the wildtype enzyme.

Seven mutations could be associated with an attenuated or

intermediate phenotype, due to low evolutionary conser-

vation, conservativeness residue changes, values of mini-

mization energy close to the wildtype enzyme, and surface

localization.

Conclusions

In summary, we have shown for the first time a docking

evaluation of natural and artificial ligands for human

GALNS enzyme, suggesting the interaction of Asp39,

Asp40, FGly79 (Cys79), Arg83, Tyr108, His142, His236,

Asp288, Asn289, and Lys310. This knowledge could pro-

vide the basis for the search of pharmacological chaperones

for Morquio A disease. Furthermore, combination of sev-

eral bioinformatics approaches allowed to explain 90 % of

missense mutations with a reported phenotype, and the

prediction of the phenotype for 21 missense mutations for

which the phenotype information was not available in the

reported literature. In most cases, severe mutations were

associated with changes of higly to completely conserved

residues, non-conservativeness changes, and low values of

Atomic Accessible Surface Area. On the other hand,

attenuated mutations seem to be associated with none to

Fig. 6 Phylogenetic trees for

GALNS from several species

(a) and for human sulfatases (b).

Sequences were aligned by

using Muscle and phylogenetic

tree was generated by using

MEGA5. Parenthesis numbers

represent GenBank and Uniprot

accession numbers for a and b,

respectively

7082 Mol Biol Rep (2014) 41:7073–7088

123

Ta

ble

3A

nal

ysi

so

fm

isse

nse

mu

tati

on

saf

fect

ing

GA

LN

Sen

zym

ew

ith

rep

ort

edp

hen

oty

pe

Muta

tion

aE

volu

tionar

yco

nse

rvat

ion

Res

ide

chan

ge

Conse

rvat

iven

ess

Posi

tion

Poly

phen

2A

tom

ic

acce

ssib

le

surf

ace

area

(A2)d

Ener

gy

Min

imiz

atio

n

(kJ/

mol)

Rep

ort

ed

phen

oty

pee

GA

LN

Sb

Sulf

atas

esc

Wil

dty

pe

-12,4

20

Norm

al

M1V

(CM

042053)

Com

ple

tely

–C

onse

rvat

iven

ess

Surf

ace

Ben

ign

––

Sev

ere

L15M

(CM

054751)

––

Conse

rvat

iven

ess

Surf

ace

Ben

ign

––

Att

enuat

ed

G23R

(CM

042054)

––

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

––

Att

enuat

ed

L36P

(CM

054746)

Inte

rmed

iate

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

18

Att

enuat

ed

M41L

(CM

041769)

Com

ple

tely

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.2

-12,4

18

Att

enuat

ed

G42E

(CM

054752)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-11,4

58

Sev

ere

G47R

(CM

970574)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,6

70

Sev

ere

D60N

(CM

970575)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly7.5

-12,5

78

Att

enuat

ed

R61W

(CM

054740)

Inte

rmed

iate

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly17

-12,1

82

Att

enuat

ed

G66R

(CM

061006)

Hig

hly

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,2

96

Sev

ere

F69

V(C

M970576)

Hig

hly

Hig

hly

Conse

rvat

iven

ess

Surf

ace

Poss

ibly

0-

12,3

86

Sev

ere

A75G

Hig

hly

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

oss

ibly

–-

12,3

38

Sev

ere

P77R

(CM

950524)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,6

63

Sev

ere

C79Y

(CM

045155)

Com

ple

tely

Com

ple

tely

Conse

rvat

iven

ess

Cat

alyti

cP

robab

ly1.3

-11,9

94

Sev

ere

S80L

(CM

970577)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Cat

alyti

cP

robab

ly0

-11,3

70

Sev

ere

R90W

(CM

950525)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.4

-10,9

83

Sev

ere

R94C

(CM

950527)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.2

-12,1

37

Att

enuat

ed

R94G

(CM

950526)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.2

-12,0

49

Att

enuat

ed

R94L

(CM

054741)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.2

-12,1

57

Sev

ere

G96C

(CM

970578)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.1

-11,7

73

Sev

ere

G96V

(CM

950528)

Com

ple

tely

Hig

hly

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.1

-11,2

59

Sev

ere

F97V

(CM

960687)

Com

ple

tely

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,3

92

Att

enuat

ed

Q111R

(CM

970579)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.7

-12,5

44

Att

enuat

ed

I113F

(CM

950529)

Com

ple

tely

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-11,6

76

Sev

ere

G116S

(CM

054742)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,4

78

Sev

ere

P125L

(CM

970581)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-11,2

31

Sev

ere

S135R

(CM

980814)

Non-c

onse

rved

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Poss

ibly

0.2

-11,9

34

Sev

ere

V138A

(CM

950530)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,4

14

Att

enuat

ed

G139S

(CM

970582)

Com

ple

tely

Com

ple

tely

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.1

-11,8

51

Sev

ere

W141R

(CM

970583)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,5

69

Sev

ere

W141C

(CM

045156)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,2

72

Sev

ere

H142R

Com

ple

tely

Hig

hly

Conse

rvat

iven

ess

Cat

alyti

cP

robab

ly0.7

-12,6

24

Sev

ere

H150Y

(CM

042056)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly1.9

-11,8

47

Att

enuat

ed

P151S

(CM

950533)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.2

-12,4

48

Sev

ere

P151L

(CM

950532)

Com

ple

tely

Hig

hly

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.2

-12,2

77

Sev

ere

G155R

(CM

970584)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly2.5

-12,5

54

Sev

ere

Mol Biol Rep (2014) 41:7073–7088 7083

123

Ta

ble

3co

nti

nu

ed

Muta

tion

aE

volu

tionar

yco

nse

rvat

ion

Res

ide

chan

ge

Conse

rvat

iven

ess

Posi

tion

Poly

phen

2A

tom

ic

acce

ssib

le

surf

ace

area

(A2)d

Ener

gy

Min

imiz

atio

n

(kJ/

mol)

Rep

ort

ed

phen

oty

pee

GA

LN

Sb

Sulf

atas

esc

G155E

(CM

041770)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly2.5

-12,3

45

Sev

ere

F156C

(CM

980815)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

48

Sev

ere

F156S

(CM

970585)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

65

Att

enuat

ed

S162F

(CM

970586)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.1

-11,8

05

Sev

ere

P163H

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.6

-12,3

77

Att

enuat

ed

C165Y

Com

ple

tely

Low

Conse

rvat

iven

ess

Cat

alyti

cP

robab

ly–

-11,4

58

Sev

ere

H166Q

(CM

970587)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Cat

alyti

cP

robab

ly0

-12,5

59

Sev

ere

F167

V(C

M054747)

Hig

hly

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

oss

ibly

15.2

-12,3

96

Att

enuat

ed

6168R

(CM

970588)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly1.6

-12,2

60

Sev

ere

D171A

(CM

001701)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly16.7

-12,4

16

Att

enuat

ed

P179S

(CM

023382)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,4

32

Sev

ere

P179L

(CM

980816)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,4

41

Sev

ere

P179H

(CM

970589)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,4

61

Sev

ere

E185G

(CM

970590)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Poss

ibly

17.9

-12,3

00

Sev

ere

N204

K(C

M920291

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

6-

12,2

45

Att

enuat

ed

W230G

(CM

950534)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

01

Sev

ere

D233N

(CM

041771)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.3

-12,5

91

Sev

ere

H236D

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Cat

alyti

cP

robab

ly4.7

-12,3

69

Att

enuat

ed

V239F

(CM

054743)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-11,4

17

Sev

ere

G247D

(CM

970591)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

18

-12,5

03

Sev

ere

R253W

(CM

001702)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly6.6

-12,1

93

Att

enuat

ed

A257T

(CM

980817)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

40

Sev

ere

R259Q

(CM

970592)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly9.8

-12,3

33

Att

enuat

ed

F284V

(CM

980818)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

0.2

-12,4

01

Att

enuat

ed

S287L

(CM

970593)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-11,1

94

Sev

ere

N289S

Com

ple

tely

Hig

hly

Conse

rvat

iven

ess

Cat

alyti

cP

robab

ly0

-12,2

36

Att

enuat

ed

G290S

(CM

970594)

Com

ple

tely

Com

ple

tely

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,4

23

Sev

ere

A291T

(CM

950535)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.4

-12,2

68

Sev

ere

A291D

(CM

970595)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.4

-12,3

49

Att

enuat

ed

S295F

(CM

970596)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.5

-12,3

13

Att

enuat

ed

G301C

(CM

970597)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly3.7

-12,0

91

Sev

ere

L307P

(CM

045154)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.4

-12,4

20

Sev

ere

G309R

(CM

970598)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.2

-8,5

89

Sev

ere

K310N

(CM

054737)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Cat

alyti

cP

robab

ly4.5

-12,5

69

Att

enuat

ed

T312S

(CM

980819)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

0-

12,4

95

Att

enuat

ed

M318R

(CM

950536)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0

-12,6

67

Sev

ere

A324E

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

78

Att

enuat

ed

7084 Mol Biol Rep (2014) 41:7073–7088

123

Ta

ble

3co

nti

nu

ed

Muta

tion

aE

volu

tionar

yco

nse

rvat

ion

Res

ide

chan

ge

Conse

rvat

iven

ess

Posi

tion

Poly

phen

2A

tom

ic

acce

ssib

le

surf

ace

area

(A2)d

Ener

gy

Min

imiz

atio

n

(kJ/

mol)

Rep

ort

ed

phen

oty

pee

GA

LN

Sb

Sulf

atas

esc

G340D

(CM

041772)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.4

-12,4

54

Sev

ere

S341R

(CM

045152)

Hig

hly

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Poss

ibly

0.8

-12,3

23

Sev

ere

M343R

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly1.6

-12,5

13

Sev

ere

M343L

(CM

950537)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

1.6

-12,4

08

Sev

ere

D344N

(CM

950538)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.1

-12,5

87

Sev

ere

D344E

(CM

970600)

Com

ple

tely

Hig

hly

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.1

-12,3

82

Sev

ere

L345P

(CM

041773)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0.7

-12,1

58

Sev

ere

F346L

(CM

950539)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

83

Sev

ere

A351V

(CM

970601)

Non-c

onse

rved

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

5.6

-12,4

02

Sev

ere

L352P

(CM

041774)

Hig

hly

Hig

hly

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly2

-11,6

55

Att

enuat

ed

P357L

(CM

042058)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

8.1

-12,4

00

Sev

ere

R361G

(CM

970602)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly6.6

-12,0

65

Sev

ere

L369P

(CM

054744)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Pro

bab

ly3.6

-11,7

67

Sev

ere

R376Q

(CM

980820)

Non-c

onse

rved

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

16.6

-12,3

44

Sev

ere

R380T

(CM

054738)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly1.2

-12,1

80

Sev

ere

R380S

(CM

054739)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly1.2

-12,1

77

Att

enuat

ed

R386C

(CM

950540)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly1.1

-12,1

64

Sev

ere

R386H

(CM

045153)

Com

ple

tely

Low

Conse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly1.1

-12,1

75

Sev

ere

D388N

(CM

042060)

Non-c

onse

rved

Low

Non-c

onse

rvat

iven

ess

Act

ive

Cav

ity

Ben

ign

5.1

-12,5

88

Att

enuat

ed

M391V

(CM

970603)

Com

ple

tely

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

0-

12,4

33

Att

enuat

ed

L395

V(C

M980821)

Inte

rmed

iate

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

4.1

-12,4

09

Sev

ere

H398D

(CM

041775)

Non-c

onse

rved

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

2.5

-12,3

57

Att

enuat

ed

N407H

(CM

970605)

Com

ple

tely

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yP

robab

ly0.4

-12,2

04

Sev

ere

W409S

(CM

970606)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Act

ive

cavit

yB

enig

n7.5

-12,3

80

Att

enuat

ed

E450

V(C

M950541)

Com

ple

tely

Hig

hly

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly0

-12,3

86

Sev

ere

F452I

(CM

054753)

Inte

rmed

iate

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Ben

ign

7.7

-12,4

26

Sev

ere

F452L

Inte

rmed

iate

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

7.7

-12,4

42

Sev

ere

P484S

(CM

054750)

Hig

hly

Low

Non-c

onse

rvat

iven

ess

Surf

ace

Pro

bab

ly4.1

-12,4

52

Att

enuat

ed

N487S

(CM

950543)

Hig

hly

Hig

hly

Conse

rvat

iven

ess

Surf

ace

Ben

ign

6.9

-12,2

49

Sev

ere

M494V

(CM

970607)

Hig

hly

Low

Conse

rvat

iven

ess

Surf

ace

Ben

ign

1.8

-12,2

32

Sev

ere

aP

aren

thes

isnum

ber

sre

pre

sent

HG

MD

acce

ssio

nnum

ber

sb

Com

ple

tely

conse

rved

:20

spec

ies;

Hig

hly

conse

rved

:15–19

spec

ies;

Inte

rmed

iate

conse

rved

:9–14

spec

ies;

and

non-c

onse

rved

:\9

spec

ies

cC

om

ple

tely

conse

rved

:17

sulf

atas

es;

Hig

hly

conse

rved

:9–16

sulf

atas

es;

low

conse

rved

:\8

sulf

atas

esd

Rep

ort

edby

Riv

era-

Colo

net

al.

[26

]e

Ref

eren

ces

[16,

26

,37

,38

]

Mol Biol Rep (2014) 41:7073–7088 7085

123

Ta

ble

4P

hen

oty

pe

pre

dic

tio

nfo

rm

isse

nse

mu

tati

on

saf

fect

ing

GA

LN

Sen

zym

ew

ith

ou

tre

po

rted

ph

eno

typ

e

Mu

tati

on

aE

vo

luti

on

ary

con

serv

atio

nR

esid

e

chan

ge

con

serv

ativ

enes

s

Po

siti

on

Po

lyp

hen

2A

tom

ic

acce

ssib

le

surf

ace

area

(A2)d

En

erg

y

Min

imiz

atio

n

(kJ/

mo

l)

Pre

dic

ted

ph

eno

typ

eG

AL

NS

bS

ulf

atas

esc

S5

3F

(CM

05

47

55

)H

igh

lyL

ow

No

n-c

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly3

.8-

12

,40

7S

ever

e

S7

4F

Inte

rmed

iate

Lo

wN

on

-co

nse

rvat

iven

ess

Su

rfac

eP

rob

ably

–-

11

,74

9S

ever

e

T8

8I

Hig

hly

Hig

hly

No

n-c

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly0

-1

2,3

65

Sev

ere

A1

07

T(C

M0

42

05

5)

Hig

hly

Lo

wN

on

-co

nse

rvat

iven

ess

Act

ive

cav

ity

Pro

bab

ly0

-1

1,9

75

Sev

ere

E1

21

DC

om

ple

tely

Hig

hly

Co

nse

rvat

iven

ess

Su

rfac

eP

rob

ably

–-

12

,38

0A

tten

uat

ed

N1

64

T(C

M0

45

15

1)

Co

mp

lete

lyL

ow

Co

nse

rvat

iven

ess

Act

ive

cav

ity

Pro

bab

ly0

.8-

12

,28

0S

ever

e

A2

03

V(C

M0

54

74

9)

Inte

rmed

iate

Lo

wC

on

serv

ativ

enes

sS

urf

ace

Ben

ign

0-

12

,40

8A

tten

uat

ed

Y2

54

CC

om

ple

tely

Hig

hly

Co

nse

rvat

iven

ess

Act

ive

cav

ity

Pro

bab

ly–

-1

2,3

57

Sev

ere

E2

60

KC

om

ple

tely

Hig

hly

No

n-c

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly–

-1

2,3

24

Sev

ere

E2

60

D(C

M0

54

74

8)

Co

mp

lete

lyH

igh

lyC

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly0

-1

2,4

19

Att

enu

ated

T3

12

AC

om

ple

tely

Lo

wN

on

-co

nse

rvat

iven

ess

Su

rfac

eP

rob

ably

0-

12

,50

2S

ever

e

G3

16

VC

om

ple

tely

Hig

hly

Co

nse

rvat

iven

ess

Act

ive

cav

ity

Pro

bab

ly0

.1-

11

,05

7S

ever

e

W3

25

C(C

M0

54

75

6)

Co

mp

lete

lyL

ow

No

n-c

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly0

-1

2,3

43

Sev

ere

L3

66

FH

igh

lyL

ow

Co

nse

rvat

iven

ess

Su

rfac

eB

enig

n7

.1-

12

,41

4A

tten

uat

ed

A3

92

V(C

M0

42

06

1)

Co

mp

lete

lyL

ow

Co

nse

rvat

iven

ess

Su

rfac

eP

rob

ably

0-

12

,39

5S

ever

e

T3

94

PIn

term

edia

teL

ow

No

n-c

on

serv

ativ

enes

sS

urf

ace

Po

ssib

ly–

-1

2,3

26

Sev

ere

L3

95

P(C

M9

70

60

4)

Inte

rmed

iate

Lo

wC

on

serv

ativ

enes

sS

urf

ace

Ben

ign

4.1

-1

2,3

49

Att

enu

ated

H4

01

Y(C

M0

54

75

4)

Co

mp

lete

lyL

ow

No

n-c

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly0

.1-

11

,43

0S

ever

e

S4

70

P(C

M0

54

74

5)

No

n-c

on

serv

edL

ow

No

n-c

on

serv

ativ

enes

sS

urf

ace

Ben

ign

24

.3-

11

,54

8A

tten

uat

ed

V4

88

M(C

M9

50

54

4)

Hig

hly

Lo

wC

on

serv

ativ

enes

sS

urf

ace

Ben

ign

17

.5-

12

,38

8A

tten

uat

ed

N4

95

YC

om

ple

tely

Lo

wC

on

serv

ativ

enes

sS

urf

ace

Pro

bab

ly–

-1

1,3

06

Sev

ere

aP

aren

thes

isn

um

ber

sre

pre

sen

tH

GM

Dac

cess

ion

nu

mb

ers

bC

om

ple

tely

con

serv

ed:

20

spec

ies;

Hig

hly

con

serv

ed:

15

–1

9sp

ecie

s;In

term

edia

teco

nse

rved

:9

–1

4sp

ecie

s;an

dn

on

-co

nse

rved

:\9

spec

ies

cC

om

ple

tely

con

serv

ed:

17

sulf

atas

es;

Hig

hly

con

serv

ed:

9–

16

sulf

atas

es;

low

con

serv

ed:\

8su

lfat

ases

dR

epo

rted

by

Riv

era-

Co

lon

etal

.[2

6]

7086 Mol Biol Rep (2014) 41:7073–7088

123

intermediate amino acid conservation, conservativeness

changes, surface position, high values of Atomic Accessi-

ble Surface Area, and energy minimization close or lower

than that of the wildtype enzyme. However, several factors

could limit the phenotype-genotype correlation, such as the

subjective classification of patients (clinical and biochem-

ical), association of GALNS with the Multienzyme Lyso-

somal Complex of b-galactosidase, cathepsin A, and

neuraminidase [25]; or epigenetic factors. Furthermore,

future anlaysis should consider the relation with the levels

of urine and blood keratin and/or chondroitin sulfate [8].

We consider that this approach can be extrapolated for the

study of phenotype–genotype correlation for other lyso-

somal storage diseases, which could have important

implications in the understanding of the disease, and the

design of new therapeutic strategies.

Acknowledgments This work was supported in part by The

Administrative Department of Science, Technology and Innovation

COLCIENCIAS (Colombia) grant 120356933205 (ID PPTA 5174) and

Pontificia Universidad Javeriana (PPTA 5596). Alexander Rodriguez-

Lopez recived a PhD scholarship from Pontificia Universidad Javeri-

ana. We thank Alexander Herrera at the High Performance Computing

Center—ZINE—of Pontificia Universidad Javeriana, for his assistance

during docking and molecular dynamics simulations.

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