Glycobiology Advance Access published May 28, 2015

1 Human Sialic Acid Acetyl Esterase (SIAE): towards a better understanding of a puzzling enzyme

Flavia Orizio1, Eufemia Damiati2, Edoardo Giacopuzzi2, Giuliana Benaglia1, Stefano Pianta1,#, Roland Schauer3, Reinhard Schwartz-Albiez4, Giuseppe Borsani2, Roberto Bresciani1, Eugenio Monti*,1 1

Unit of Biotechnology, Department of Molecular and Translational Medicine (DMTM),

2

Unit of Biology and Genetics, Department of Molecular and Translational Medicine

(DMTM), University of Brescia, Viale Europa 11, 25123 Brescia, Italy 3

Institute of Biochemistry, Christian-Albrechts University, Otto-Hahn-Platz 9, 24118 Kiel,

Germany 4

Department of Translational Immunology, Unit of Glycoimmunology, German Cancer

Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany *

Corresponding author: Tel: +390303717544; Fax: +390303701157; e-mail:

[email protected]; #

Present address: Centro di Ricerca E. Menni (CREM), Fondazione Poliambulanza-Istituto

Ospedaliero, Via Bissolati, 57, 25124 Brescia, Italy

© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

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University of Brescia, Viale Europa 11, 25123 Brescia, Italy

2 ABSTRACT Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the “long” isoform, originally

approach we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4 - 8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

Keywords Sialic acid acetyl esterase / SIAE / Neu5Ac / O-acetylation / in silico characterization / phylogenesis / 3D model / subcellular localization.

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referred to lysosomal sialic acid esterase (Lse), is detected. Using the homology modeling

3 INTRODUCTION Sialic acids as terminal sugars of N- and O-glycosylated macromolecules are decisive for the functions of glycoconjugates both in soluble form and integrated into cell membranes (Schauer, R. 2009, Varki, A. 2008). A wide range of structural diversity characterizes sialic acids, with O-acetylation at the hydroxyl group of carbon C9 as the most frequent modification. Functions and biosynthesis of O-acetylated sialic acids have been recently reviewed (Mandal, C., Schwartz-Albiez, R., et al. 2012) and among the great variety of

system (Schauer, R., Srinivasan, G.V., et al. 2011), in particular for human B cell differentiation (Erdmann, M., Wipfler, D., et al. 2006, Wipfler, D., Srinivasan, G.V., et al. 2011) and autoimmunity (Pillai, S., Cariappa, A., et al. 2009). In this perspective the balance between O-acetylation and de-O-acetylation of sialic acid as an on/off switch mechanism is able to regulate distinct processes such as apoptosis (Kniep, B., Kniep, E., et al. 2006, Malisan, F., Franchi, L., et al. 2002, Mukherjee, K., Chava, A.K., et al. 2008). The two enzymes responsible for acetylation and de-acetylation have been described as sialate Oacetyltransferase (SOAT) and sialate O-acetylesterase (SIAE), respectively (Mandal, C., Schwartz-Albiez, R., et al. 2012). Recently, the Cas1 protein has been reported as a possible candidate for sialic acid specific O-acetytransferase of sialic acids (Arming, S., Wipfler, D., et al. 2011). The first description of an esterase acting on 4-O and 9-O acetyl groups of free or bound sialic acid dates back to 1983 (Shukla, A.K. and Schauer, R. 1983) and from 1986 onwards (Varki, A., Muchmore, E., et al. 1986) several studies have been published on Sialic Acid Acetyl Esterase (SIAE) (Mandal, C., Schwartz-Albiez, R., et al. 2012). SIAE (sialate Oacetylesterase, EC 3.1.1.53) is largely diffuse in nature and several forms of the enzyme with regards to its origin are reported (Enzyme Database-BRENDA, www.brenda-enzymes.info/) ranging from viruses and bacteria to vertebrates. A functional link between SIAE activity and

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phenomena involving these modified sialic acids, several have been described for the immune

4 B cell development, signaling, and immunological tolerance has recently been described in rodents (Cariappa, A., Takematsu, H., et al. 2009). Indeed, engineered mice with catalytically deficient SIAE, showed enhanced B cell receptor (BCR) activation, defects in peripheral B cell development, presence of autoantibodies against chromatin and developed glomerular deposits of immune-complexes. At the molecular level, this effect could be mediated by the 9-O-acetylation state of sialic acid, which in turn regulates the function of CD22/Siglec-2. Actually, SIAE deacetylates (2-6) linked sialic acid on N-linked glycans of BCR, allowing

Shock, A., et al. 2012, Pillai, S., Netravali, I.A., et al. 2012). Starting from 2010, functionally defective SIAE variants have been associated to susceptibility to autoimmune disorders in human subjects (Hirschfield, G.M., Xie, G., et al. 2012, Surolia, I., Pirnie, S.P., et al. 2010). However, a subsequent large study on approximately 67,000 subjects failed to replicate the association with autoimmune and chronic immune diseases (Hunt, K.A., Smyth, D.J., et al. 2012), pointing out the challenges of associating low frequency variants with complex traits and disease. Recently, catalytically defective rare variants of SIAE have been again associated with autoimmunity, reopening the debate about the role played by this enzyme in immunological response (Chellappa, V., Taylor, K.N., et al. 2013). From the very beginning SIAE has been described as an enzyme presenting two (iso-)forms, the cytosolic (Cse) and the lysosomal/membrane-associated (Lse) enzyme (Butor, C., Diaz, S., et al. 1993, Butor, C., Griffiths, G., et al. 1995, Diaz, S., Higa, H.H., et al. 1989, Higa, H.H., Diaz, S., et al. 1987, Varki, A., Muchmore, E., et al. 1986). Subsequent studies on the enzyme resulted in contrasting findings (Cariappa, A., Takematsu, H., et al. 2009, Guimaraes, M.J., Bazan, J.F., et al. 1996, Stoddart, A., Zhang, Y., et al. 1996) and several aspects of SIAE biology remain still unsolved. For example, different molecular weight subunits of the cytosolic enzyme, originated by proteolytic cleavage of the lysosomal protein during maturation in the

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the interaction with CD22, that functions in vivo as an inhibitor of BCR signaling (Dorner, T.,

5 endosome/lysosome compartment, have been reported for SIAE (Takematsu, H., Diaz, S., et al. 1999) as well as differential splicing events have been described for SIAE (Stoddart, A., Zhang, Y., et al. 1996). Also the functional impact of the subcellular/extracellular localization of the protein(s) remains unclear (Mandal, C., Schwartz-Albiez, R., et al. 2012). In 2004, the cDNA encoding for the human SIAE has been isolated by using differential display RT-PCR from testis, revealing a high degree of sequence identity to the mouse enzyme and an apparent lysosomal localization of the protein in a human pancreatic adenocarcinoma cell line (Zhu,

orthologs have been identified from various organisms, a detailed phylogenetic analysis of this enzyme(s) has never been carried out. Moreover, based on the presence of conserved residues and on functional data, including the use of inhibitors (Schauer, R., Reuter, G., et al. 1989), SIAE enzyme(s) are classified as serine esterases, belonging to a super family of hydrolases with more than 7,000 members (see for details UniProt KW-0719). Surprisingly, no structural data are available for SIAE enzymes from higher organisms, whereas detailed studies have been carried out on microbial esterases acting on sugar residues (Pfeffer, J.M., Weadge, J.T., et al. 2013), on a SIAE from Escherichia coli (Rangarajan, E.S., Ruane, K.M., et al. 2011) and further extensive studies on viral SIAE like from influenza C-, corona- and torovirus (de Groot, R.J. 2006, Herrler, G. and Klenk, H.D. 1991). Trying to get new insights on human SIAE biology and in order to clarify its debated molecular properties we conducted a multidisciplinary study on this enzyme. We combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells revealing new details on the biology of SIAE enzyme.

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H., Chan, H.C., et al. 2004). Although several other DNA sequences encoding for SIAE

6 RESULTS

Characterization of the human SIAE gene

The human SIAE gene is located on chromosome 11 at position 11q24.2 (Zhu, H., Chan, H.C., et al. 2004). As summarized in the introduction, previous studies report that it encodes two different transcript isoforms named variant 1 and 2 also listed in the Gene database at

Browser. Variant 1 (RefSeq NM_170601) spans 10 exons and represents the shorter transcript but encodes the longer isoform 1 of the protein, also referred to as lysosomal sialic acid esterase (Lse). Variant 2 (RefSeq NM_001199922.1) spans 12 exons that differs in the 5' UTR, and its first coding exon corresponds to the second of variant 1. The resulting isoform 2 of the protein has a different internal N-terminus and is also referred to as the cytosolic sialic acid esterase (Cse). For these reasons, Lse is 523 aa long (RefSeq NP_733746), while Cse has 488 aa (RefSeq NP_001186851) lacking the first 35 aa of the lysosomal form. A bioinformatics analysis revealed the presence of 78 Expressed Sequence Tags (ESTs) generated from various human tissues and cell types supporting the SIAE transcript variant 1, while no ESTs corresponding to SIAE transcript variant 2 were found (Supplementary figure 2A). The Ensembl genome browser reports only two SIAE transcripts with protein coding capabilities: SIAE-001, corresponding to the Lse isoform (RefSeq NM_170601, NP_733746), and SIAE-201 which is identical to the previous one with the exception that the second in frame ATG codon is considered as translation starting site. Although this would result in a 488 aa polypeptide identical to the Cse encoded by the above mentioned variant 2 (RefSeq NM_001199922.1, NP_001186851), a bioinformatic analysis carried out with algorithms for

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NCBI. Supplementary figure 1 shows the two variants as displayed in the UCSC Genome

7 identifying the initiation codons in cDNA sequences strongly support the first in frame ATG as the most reliable translation start site. No protein coding transcript with an alternative 5’ exon corresponding to the RefSeq NM_001199922.1 cytosolic SIAE isoform is listed by Ensembl. Furthermore, the analysis of RNASeq data from Illumina’s Human BodyMap 2.0 project indicates that in 15 out of the 16 human tissues analyzed exon-exon boundaries correspond to variant 1, while there is no transcript model supporting variant 2 (Supplementary Figure 2B).

isoforms that differ in length likely as a consequence of an alternate promoter (Takematsu, H., Diaz, S., et al. 1999). An in silico analysis showed that also in mice, only the Lse transcript variant is present in the ESTs database with 55 sequences (data not shown). Furthermore, NCBI Gene database reports as gene product a single reference sequence (RefSeq) of 541 aa (NP_035864), corresponding to Lse. It has to be noted that 5 mouse ESTs (4 from whole joint and one from corpora quadrigemina) are present in dbEST having an alternative 5’ exon compared to the Siae Lse transcript. These ESTs correspond to a transcript variant named X1 encoding a 553 aa polypeptide (XP_006510217), with the first 35 differing from the Lse isoform. Overall, these data indicate that in adult human and mouse tissues expression of SIAE transcripts is almost exclusively limited to the lysosomal SIAE isoform. RNA-Seq data generated from 27 human tissues and made available through the Expression Atlas database have been used to infer the expression level of the SIAE gene. Supplementary figure 3 indicates that the gene is expressed in all tissues tested although at different levels. The highest levels of expression have been observed in colon, kidney and thyroid gland.

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Previous studies indicate that the Siae mouse gene behaves quite similarly, encoding for two

8 In silico characterization of SIAE protein

Genome sequencing projects recently completed on several organisms allow the retrieval of valuable information about molecular phylogenesis of the gene of interest (Genome, K.C.o.S. 2009, Mulder, N.J., Kersey, P., et al. 2008). This allowed us to carry out the first phylogenetic analysis of SIAE, pointing out several interesting features of this enzyme. To identify highly conserved motifs and residues, we used MUSCLE to generate a multiple

(Supplementary figures 4 and 5, Supplementary Table 1). According to the criteria defined in Materials and Methods, we were able to identify 11 highly conserved sequence blocks (Figure 1). Among them we also identified 4 sequence blocks characterizing family II of SGNH hydrolase superfamily (esterase blocks) and the 2 essential residues required for catalysis, represented by the fully conserved amino acids S127 and H377. The predicted N-terminal signal peptide is highly conserved in vertebrates, with the exception of Taeniopygia. guttata and Xenopus. laevis (Supplementary figure 5A). To complete the in silico characterization of the human SIAE, we also investigated the presence of N- and O-glycosylation sites, as well as phosphorylation sites in the protein sequence. Using the tools described in Materials and Methods we predicted 6 Nglycosylation, 10 O-glycosylation (4 with high confidence) and 24 phosphorylation sites (Table 1, more details in Supplementary Table 2-4). We investigated the location of these sites are located in the predicted protein structure. The sites T58, T73 and N107 are in the Nterminal portion, not included in the final structure. The other 5 N-glycosylation sites and 7 O-glycosylation sites are, as expected, exposed on the protein surface, while T383 is located inside the predicted structure (Supplementary figure 7). Analysis of the conservation at these positions on the primary structure identified 2 glycosylation sites (N138 and S177) highly

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sequence alignment with sequences from 32 protein orthologs of human SIAE

9 conserved through Metazoa, and 5 phosphorylation sites (S30, S227, S228, T221, S255) highly conserved in all the 32 protein sequences used in our multiple alignment (Figure 2). Using subfamily LOGO to asses for conservation in specific subgroups, we identified also 2 potential glycosylation sites conserved only in Vertebrata and 3 conserved only in Mammalia, as well as 5 phosphorylation sites conserved only in Metazoa and 5 only in Mammals (Figure 2). In silico predictions revealed the presence of an N-terminal signal peptide in human SIAE corresponding to amino acids M1-A18. This result is in agreement with data from the Signal

(M1-A23). Secretome 2.0 prediction showed that human SIAE has a high score (NN-score 0.78) as a secreted protein. Interestingly, an amino acid stretch of variable length (22-27 residues) typical of Muroidea has been found between conserved block 5 and 6 (Supplementary figure 5, and Supplementary figure 8). Localization of these residues in the predicted model indicates that they belong to the loop connecting the -strand 4 and the -helix C (see for details Figure 3A and Supplementary figure 8B). It is tempting to speculate such an insertion could confer some peculiar biochemical feature(s) to SIAE of this large superfamily of Rodents (Jiang, H. and Blouin, C. 2007). Considering the relevance given by some studies to SIAE genetic variants and their association to immune disease (Pillai, S. 2013), we searched the Exome Aggregation Consortium (ExAC) data set of 60,706 unrelated individuals and produced a catalog (Supplementary table 5) of the genetic variability of SIAE gene in humans focused on single nucletide variants (SNVs) affecting residues involved in either post-translational modification (Table 1) or catalytic activity (S127, T132 and H377).

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Peptide Database (http://www.signalpeptide.de/), reporting a slightly longer signal peptide

10 SIAE structure prediction

Homology modeling based on the structure of known esterases from Escherichia coli (3PT5), Clostridium acetobutylicum (1ZMB) and Arabidopsis thaliana (2APJ) allowed the generation of a complete 3D model for the human SIAE protein (Supplementary figure 6). The Nterminal (M1-V112) and C-terminal (L402-K523) regions of the human protein were difficult to align to the template structure, resulting in long, mainly disorganized loops and were thus

model (Figure 3A) shows the presence of an // structure typical of SGNH-hydrolase family, composed by 5 parallel and 1 anti-parallel sets of β-sheets and 12 α-helices (Molgaard, A., Kauppinen, S., et al. 2000). The parallel β-sheets are buried in the protein core, surrounded by the -helical structures (Figure 3B and C), which contribute to most of the protein surface. The two residues S127 and H377, together with the 4 esterase blocks of conserved residues (Molgaard, A., Kauppinen, S., et al. 2000) are placed on the predicted model to assess the position of the putative active site. Together with the conserved block 5 (G219-S228), these portions of the protein constitute a surface suitable to represent the catalytic crevice (Figure 3, 4 A and B). Automated blind docking of 9-O-Acetyl-Neu5Ac substrate on the structural model of human SIAE placed the molecule in the predicted active site as one of the best hits (estimated G = 6.59 kcal/mol), further supporting the predicted conformation. The distance between the C9O bond of the substrate molecule and the two catalytic residues S127 and H377 is 3.8 and 4.3 Å, respectively Moreover, using the generated molecular complex, we were able to identify the residues possibly involved in interactions with the substrates (indicated with + in Figure 1): T132, Q135, Q175, W176, S177, K178, T180, M192, W218, G219, G220, S332. Among these, the residues T132 and S332 are placed near the essential H377 and represent the best

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removed from subsequent analysis of the predicted structure. The topology of the predicted

11 candidates as possible third element involved in the esterase catalytic triad. Mutagenesis experiments (see below) demonstrated that the three residues T132, S332 and H377 compose the actual catalytic triad. The architecture of the predicted active site of human SIAE is shown in Figure 4.

Cloning and expression of SIAE in COS7 cells

so-called lysosomal SIAE or Lse is by large the prevalent isoform expressed in all tissues investigated. We thus cloned the lysosomal SIAE coding region (1572bp) into the expression vector pMT21 allowing the expression of a myc-tagged fusion protein. The resulting construct (pMT21-SIAE) was used to follow expression of the protein in transiently transfected COS7 cells for up to 48h by means of enzymatic activity and Western-blot analysis. In cell extracts derived from pMT21-SIAE specific COS7 transfectants, an increase in SIAE activity was already detected after 24h transfection and after 48h the enzymatic activity was increased roughly 5 to 6-fold compared to non-transfected cells, indicating an enzymatically active overexpressed protein (Table 2). Of note, in the conditioned culture media of cells transfected for 24h, only a negligible increase in enzymatic activity was detected, whereas after 48h expression almost a 5-fold increase was observed with respect to the media of non-transfected cells (Table 2). Finally, using cell extracts as enzyme source, we found an in vitro alkaline pH optimum of 8.4 - 8.5, with a negligible enzyme activity at pH below 6.0 (Figure 5A). Taken together, these data indicate that after 48h expression, SIAE is released into the growth medium and that the balance between intracellular-to-extracellular activities is in favor of the secreted pool in a ratio of 1:3.5, respectively.

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As described above, the in silico analysis of various transcriptome databases indicates that the

12 Western-blot analysis confirmed the presence of SIAE, both in cellular extracts and conditioned culture media (Figure 5B). Interestingly, intracellular SIAE was detected already after 24h expression as a 62 kDa immunoreactive band, a molecular weight higher than the expected 58 kDa, together with a second and less intense band of 49 kDa and after 48h the intracellular amount of SIAE increased significantly with the same protein pattern. In order to assess whether increase in the molecular weight of SIAE could be due to glycosylation, to COS7 cells expressing the enzyme for 24h tunicamycin was added to the medium for further

cell extracts, in favor of the lower molecular weight band of 49 kDa (Figure 5C), suggesting that the increase in the molecular weight is the result of a glycosylation process. Concerning the release of SIAE from cells, after 24h expression a faint immunoreactive band of 62 kDa was detected in the growth medium and, as expected from the enzyme activity (Table 2), at 48h expression its intensity is significantly increased (Figure 5B). Compared to the 24h expression, Tunicamycin treatment resulted in the increase of the 62 kDa band in the conditioned medium, probably corresponding to release of the high molecular weight form of SIAE produced by the cells in the first 24h of expression. To better characterize synthesis and processing of newly synthesized SIAE, COS7 cells were treated with cyclohexamide under the experimental conditions described above. After a 24h treatment, the drug leads to the disappearance of the 49 kDa band in favor of the 62 kDa form, with a corresponding increase of the same band in the culture media. Overall, these data suggest that, at least in our expression system, the 58 kDa form of SIAE is rapidly proteolitically cleaved to a 49 kDa form or glycosylated to the 62 kDa form. Moreover, only the high molecular weight of the protein is secreted into the growth medium, indicating a possible trafficking of the glycosylated protein through the exocytic route.

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24 h. Tunicamycin treatment resulted in the complete disappearance of the 62 kDa band in

13 We also investigated whether glycosylation influences the enzymatic activity of SIAE. For this purpose we measured the enzyme activity both in cells extracts and medium under the experimental conditions described above. As shown in Table 2 and Figure 5B, in cell extracts SIAE specific activity correlates with the intensity of the 62 kDa band detectable in Westernblot experiments, under normal expression conditions or in presence of tunicamycin or cycloheximide. The same evidence is obtained by considering the enzyme activity released in the culture medium. To further confirm these results, cell extracts deriving from COS7 cells

conditions. As shown in Figure 5C, this treatment resulted in the disappearance of the 62 kDa band in favor of the 49 kDa band and again, the pattern of SIAE specific activity is roughly superimposable with Western-blot results. These data indicate that SIAE glycosylation is essential for its secretion into the growth medium and, as demonstrated by tunicamycin and PNGase-F treatment, influence the enzyme activity and/or stability.

Role of T132, S332 and H377 amino acid residues in determining the enzymatic activity of SIAE

In order to determine the possible role played in the catalytic activity of SIAE by the amino acid residues T132, S332 and H377 (for details see ”SIAE structure prediction” section of the Results), we generated alanine-substitution mutants in these positions by site directed mutagenesis and expressed them in COS7 cells. After 48 h expression, cells and respective media were collected and analyzed. The Western-blot analysis evidenced the presence of the 62 kDa band in both cell extracts and in the culture media, demonstrating that mutants are indeed expressed (Figure 6). The alanine substitution at position S332 did not influence

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expressing SIAE for 48h were subjected to PNGase-F treatment under non-denaturing

14 significantly the activity of the enzyme, whereas mutagenesis at position T132 and H377 abolished the enzymatic activity that resulted comparable to non-transfected cells. These data clearly demonstrate that amino acids T132 and H377 play an essential role in the catalytic activity of SIAE, defining the catalytic triad of the enzyme as composed, beside S127 (Surolia, I., Pirnie, S.P., et al. 2010), by T132 and H377.

SIAE subcellular localization

localization of the myc-tagged fusion protein expressed upon transient transfection with pMT21-SIAE of COS7 cells. Laser confocal microscopy analysis showed SIAE associated with intracellular tubular and vesicular structures (Figure 7). Co-localization experiments using PDI and GM130 as typical ER and Golgi complex markers, respectively, demonstrated the presence of the enzyme in both membranous structures (Figure 7A and B). Presence of SIAE in these subcellular compartments is in agreement with the results described above, i.e. the enzyme is a glycosylated and secreted protein. In contrast to what previously reported on lysosomal SIAE (Takematsu, H., Diaz, S., et al. 1999, Zhu, H., Chan, H.C., et al. 2004), no SIAE signal was detected in vesicular structures positive for LAMP1 (Figure 7C), a typical lysosomal marker.

DISCUSSION

Despite the interest on SIAE and its role in a variety of important biological events, several features of the enzyme remain unsolved (Mandal, C., Schwartz-Albiez, R., et al. 2012). Here we describe the characterization of the enzyme encoded by SIAE transcript variant 1

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Immunofluorescence experiments have been carried out in order to study the subcellular

15 (NM_170601.4) that, based on the literature (Zhu, H., Chan, H.C., et al. 2004), encodes for the so-called lysosomal isoform of sialic acid esterase (Lse). The in silico analysis we performed strongly indicates that this is the prevalent transcript isoform expressed in most human tissues. These data suggest that the 523 amino acids SIAE protein is responsible for the acetyl esterase activity on Neu5Ac in most tissues and cell types. Considering the number of studies on genetic variants of SIAE and their association with an increased risk for various autoimmune diseases, we also compiled a catalog of single

particular, we focused on variants of residues that are potentially relevant for the biological activity of the enzyme (i.e. post-translational modification or catalysis). Our analysis indicates that these residues present either no sequence variation in the data set analyzed of more than 60,000 individuals, or the frequency of variants is very low (below 0.001212, see Supplementary Table 5), supporting the functional role of the sites considered. We also evaluated the intolerance score of SIAE, based on an approach that assesses whether genes have relatively more or less functional genetic variation than expected based on the apparently neutral variation found in the gene (Petrovski, S., Wang, Q., et al.). Interestingly, the SIAE gene has a residual variation intolerance score (RVIS) of 0.38, which indicates the presence of an excess of common functional variation, a feature that is often shared by genes linked to immunological disorders. Phylogenetic analysis of SIAE proteins in Metazoa revealed the presence of 11 highly conserved sequence blocks (Figure 1 and Supplementary figure 5). Among them, the alignment with already characterized esterases allowed the identification of the four blocks typical of the SGNH hydrolases (Figure 1) (Molgaard, A., Kauppinen, S., et al. 2000) and of the 2 essential residues S127 and H377. The serine and histidine residues are always present in the SGNH hydrolases. The catalytic triad is completed by an acidic residue or the main-

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nucleotide variants from public repository of large next generation sequencing projects. In

16 chain carbonyl group of serine, threonine or tryptophan, that participate in the charge relay system necessary for the activation of the serine nucleophile (Akoh, C.C., Lee, G.C., et al. 2004). This third member is less conserved and varies across different SGNH hydrolases (Rangarajan, E.S., Ruane, K.M., et al. 2011, Wei, Y., Schottel, J.L., et al. 1995). Homology modeling of the human SIAE based on the known structures of esterases from Arabidopsis thaliana (Bitto, E., Bingman, C.A., et al. 2005), Escherichia coli (Rangarajan, E.S., Ruane, K.M., et al. 2011) and Clostridium acetobutilicum (PDB 1ZMB) allowed the

revealed that the human protein is composed by a core portion (aa 113-401) (Figure 3B, C) highly similar to those of other esterases, and a C-terminal domain (L402-K523) containing 3 -helices, that poorly aligns with template structures. The core structure of human SIAE is composed by 1 anti-parallel and 6 parallel β-sheets surrounded by 12 α-helices (Figure 3), a structure peculiar of SGNH hydrolase, family II (Lo, Y.C., Lin, S.C., et al. 2003, Molgaard, A., Kauppinen, S., et al. 2000), further supporting its classification in this protein family. The placement of the two essential residues S127 and H377 in the structural model is compatible with their catalytical function. These residues, together with residues from conserved block 5 (G219-S228), define a surface suitable to represent the enzyme active site (Figure 4). Molecular docking analysis using SwissDock placed 9-O-Acetyl-Neu5Ac substrate in the predicted active site as the fourth most favorable result. However, in the top 3 predicted complexes the substrate molecule interacts with the unstructured C-terminal of the human SIAE and thus the predicted active site represents the best placement in the enzyme core structure. Using PyMol to assess residues near the substrate molecule, we were able to identify 12 residues putatively involved in interactions with the substrate that could play a role in substrate specificity (marked with + in Figure 1). Overall, the predicted active site consists of a solvent-exposed shallow pocket, similar to that of NanS from E. coli

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reconstruction of the enzyme structure (Supplementary figure 6). Evaluation of this model

17 (Rangarajan, E.S., Ruane, K.M., et al. 2011) that is consistent with the binding of various complex substrates containing acetylated sialic acid (Figure 4A). We then analyzed the predicted active site to identify the actual catalytic triad. The residues S127 and H377 are fully conserved in Metazoa and the two residues T132 and S332 are placed near H377, making them good candidates to fulfill the role of the third member in the catalytic triad (Figure 4B). Mutation of the S127 residue has already been described to severely reduce SIAE enzyme activity (Surolia, I., Pirnie, S.P., et al. 2010), supporting its essential role for

putative members of the active triad and demonstrated, upon overexpression of the mutants H377A and T132A, that both are essential for enzyme activity (Figure 6). These findings define the actual catalytic triad of human SIAE to be composed by S127, T132 and H377 residues (Figure 4B). Bioinformatics analysis also predicted 6 N-glycosylation and 10 O-glycosylation sites (Table 1 and supplementary table 2-4). Glycosylation is a key modification for secreted proteins (Benham, A.M. 2012) and our data suggest that these modifications are also involved in SIAE enzymatic activity. Various glycosylation patterns have been demonstrated occurring on human SIAE (Yin, X., Bern, M., et al. 2013) and the N402 and N422 glycosylation sites have been also experimentally validated by previous studies (Chen, R., Jiang, X., et al. 2009, Tang, J., Liu, Y., et al. 2010). However, our analysis revealed that these two sites are weakly conserved in Metazoa, with the N422 conserved only in mammals (Figure 2A) and N402 only in primates. The predicted glycosylation sites N138 and S177 were fully conserved in Metazoa (Figure 2A, B) and thus represent good candidates for future studies on SIAE glycosylation. Moreover, all of the most conserved sites, as well as N402 and N422 that have been demonstrated to be glycosylated in vivo, are exposed on the protein surface in our 3D model (Supplementary figure 7), supporting both their role as potential glycosylation sites and

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catalysis. Using site-directed mutagenesis, we investigated the contribution of the other

18 the overall likelihood of the proposed model structure. To complete the in silico characterization of the protein we also predicted phosphorylation sites (Table 1 and supplementary table 4). Although the role of phosphorylation for secreted proteins is not well understood, this post-translational modification could be involved in protein secretion (Tagliabracci, V.S., Xiao, J., et al. 2013) as phosphorylated proteins are present in the secretome (Carrascal, M., Gay, M., et al. 2010). Moreover, recent studies have also demonstrated the presence of active kinases in the extracellular environment that act on

et al. 2012). Our analysis identified 5 top conserved phosphorylation sites and 5 additional sites conserved throughout Metazoa (Figure 2C). These are strong candidate sites for phosphorylation of human SIAE and could be involved in regulation of the enzyme activity and/or subcellular localization. In addition, an N-terminal signal peptide is predicted in silico and reported in the Signal Peptide Database and SecretomeP 2.0 server predicts human SIAE as a secreted protein. The expression of the myc-tagged protein chimera in fibroblast-like COS7 cells provided interesting information. First of all, using this cell line, the enzyme is to the largest extent secreted into the culture media. Accordingly, immunofluorescence staining demonstrates ER and Golgi apparatus localization, overlapping the typical secretion pathway (Figure 6). Our finding is in agreement with recent results obtained by using a (glyco-)proteomic approach on the secretome of human endothelial cells (Yin, X., Bern, M., et al. 2013), as well as with those obtained in COS7 (Guimaraes, M.J., Bazan, J.F., et al. 1996) and U2OS cells (Cariappa, A., Takematsu, H., et al. 2009) upon expression of the SIAE of mouse origin and in HEK293T cells with the enzyme of human origin (Chellappa, V., Taylor, K.N., et al. 2013). Concerning the secretion mechanism(s), SIAE has been found in urinary exosomes (Gonzales, P.A., Pisitkun, T., et al. 2009, Principe, S., Jones, E.E., et al. 2013, Prunotto, M.,

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various secreted proteins to modulate their biological activity (Tagliabracci, V.S., Engel, J.L.,

19 Farina, A., et al. 2013). In detail, expression of human SIAE in COS7 cells, inhibition of its glycosylation and the block of its synthesis demonstrated that the enzyme is either processed to a low molecular weight form, corresponding to the 49 kDa band, or is glycosylated giving rise to the biologically active form of the enzyme, corresponding to the 62 kDa band. Moreover, we demonstrated that only the glycosylated, 62 kDa high molecular weight form of SIAE is secreted into the medium and PNGase-F treatment under non-denaturing condition suggests that glycosylation represent an important step for the catalytic activity of the

SIAE glycosylation has been initially demonstrated in the mouse form (Guimaraes, M.J., Bazan, J.F., et al. 1996) and more recently in the human protein and, in particular, for N401 and N422 residues through a detailed glycoproteomic analysis of liver tissue (Chen, R., Jiang, X., et al. 2009). In the case of N401, glycosylation has been further confirmed in human hepatocellular carcinoma cell line 7703 (Tang, J., Liu, Y., et al. 2010). In this perspective, SIAE is expected to act mainly on cell surface or extracellular substrates bearing 9-Oacetylated sialic acid residues on their glycan moieties. Surprisingly and despite the literature (Butor, C., Griffiths, G., et al. 1995, Zhu, H., Chan, H.C., et al. 2004), in our experimental conditions there is no evidence of cytosolic or lysosomal localization of SIAE. Our results are in agreement with those obtained upon stable transfection of murine SIAE in human osteosarcoma cells, where an almost complete lack of lysosomal localization was observed and the protein was secreted and recovered on the cell surface (Cariappa, A., Takematsu, H., et al. 2009). In addition, the in vitro pH optimum observed, corresponding to 8.4 - 8.5, is not consistent with an activity of the enzyme within the acidic environment of lysosomes. Moreover, the absence of a typical lysosomal localization is consistent with the subcellular localization of endogenous SIAE reported in The Human Protein Atlas (Uhlen, M., Fagerberg, L., et al. 2015). Nevertheless one must consider that subcellular localization may

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enzyme.

20 be dependent on the respective cell type investigated. The SIAE expression levels and the non-synonymous common variants could somehow influence the overall behavior of the enzyme (i.e. intracellular localization or secretion into the extracellular environment). For example, a study carried out using 293T human embryonic kidney cell, BJAB and Ramos B cell lines, and peripheral blood mononuclear cells (PBMC) showed a peculiar behavior of the enzyme (Chellappa, V., Taylor, K.N., et al. 2013). Actually, the enzyme appears mainly located intracellularly, and only wild-type protein and active variants are secreted upon

detectable outside the cells. Despite some contrasting evidences (Muthusamy, B., Hanumanthu, G., et al. 2005, Polanski, M. and Anderson, N.L. 2007), the presence of SIAE has been demonstrated in human colostrum (Palmer, D.J., Kelly, V.C., et al. 2006) and plasma (Farrah, T., Deutsch, E.W., et al. 2011), and in this perspective the lack of release into the extracellular environment could reflect cellular retention due to structural defects leading to misfolding and to an inactive/less active enzyme. In COS7 cells, we observed a 3.4-fold ratio between extracellular/intracellular SIAE, supporting the notion of an enzyme acting on substrates localized in either intracellular vesicles or extracellular/cell surface environment. A recent, detailed investigation on glycosphingolipids in colorectal cancer tissues revealed a series of acetylated gangliosides that could represent possible natural substrates of the enzyme (Holst, S., Stavenhagen, K., et al. 2013). One of the most interesting target molecules for SIAE activity is the ganglioside O-acetylated GD3. Differences in acetylation state of this ganglioside are reported to influence a variety of biological effects, from apoptosis to extravasation of tumor cells, depending also on its subcellular localization (i.e. cell surface or intracellular compartments) (Schauer, R., Srinivasan, G.V., et al. 2011). Acetylated sialic acids have been found also in GM1, GD2, GD1, as well as in other gangliosides with glycans characterized by up to eight sugars bearing one or two sialic acid residues (Holst, S.,

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transfection of the corresponding cDNAs, whereas inactive or less active variants are barely

21 Stavenhagen, K., et al. 2013). Concerning possible glycoprotein substrates, acetylated sialic acid residues have been found in colon mucins, namely in the carbohydrate moieties of MUC1 and MUC2, and a decrease in their acetylation levels is found in colon carcinoma metastases (Mann, B., Klussmann, E., et al. 1997). Perhaps, the most prominent protein influenced by acetylation of sialylated glycoconjugates is the sialic acid-binding immunoglobulin-like CD22, expressed by B cells and acting as a negative regulator of B cell activation (Crispin, J.C., Hedrich, C.M., et al. 2013). Acetylation in position 9 of sialic acid

regulation of B cell response and a valuable candidate in a series of autoimmune diseases (Gan, E.H., MacArthur, K., et al. 2012, Pillai, S. 2013, Pillai, S., Netravali, I.A., et al. 2012, Varki, A. and Gagneux, P. 2012, Yamamoto, M., Iguchi, G., et al. 2014). Overall, our results describe several novel features of human SIAE, showing activity in a range of pH rather high for a typical lysosomal enzyme and supporting its localization in the ER, along the secretory pathway compartments, and mostly as a glycosylated, secreted protein. Additionally, our genomic and phylogenetic analysis, together with molecular modeling studies, provide novel insight on SIAE protein, an enzyme that acting on chemical modification such as acetylation, contributes to the fine tuning of sialic acid biology.

MATERIALS AND METHODS

In general, standard molecular biology techniques were carried out as described by Greem and Sambrook (Green, M.R. and Sambrook, J. 2012). Reagents and powders were from Sigma unless otherwise indicated.

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impairs recognition of these glycoconjugates by CD22, making SIAE a key enzyme in the

22 SIAE gene investigations

SIAE gene features have been investigated using both the UCSC (http://genome.ucsc.edu) and Ensembl (http://www.ensembl.org/) Genome Browsers using the February 2009 human reference sequence (GRCh37). Nucleotide and amino acid sequences were compared to the non-redundant sequences present at the NCBI (National Center for Biotechnology Information) using the BLAST algorithm

RNA-Seq data have been obtained from the Illumina’s Human BodyMap 2.0 project made available through Ensembl. In addition, RNA-Seq expression data on human SIAE gene have been retrieved from Expression Atlas, a database maintained by EMBL-EBI (http://www.ebi.ac.uk/gxa/home). The catalog of single nucleotide variants (SNVs) of the SIAE gene shown in Supplementary table 5 has been generated using data from the Exome Aggregation Consortium (ExAC), Cambridge, MA (http://exac.broadinstitute.org), as of February 2015.

Multiple sequence alignment and phylogenetic analysis

Human SIAE protein sequence (NP_733746.1) was used as query in DELTA-BLAST search to retrieve the putative orthologs of SIAE in 27 different organisms throughout Eukarya, plus 5 bacterial species (Supplementary Table 1). Multiple alignment of the 32 protein sequences was constructed with MUSCLE and then manually refined with JalView (Waterhouse, A.M., Procter, J.B., et al. 2009), which was also used for the calculation of conservation score at each position. Highly conserved blocks were defined as protein regions with at least 3 residues showing conservation score above 8 and with no more than 2 consecutive residues

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(Altschul, S.F., Gish, W., et al. 1990).

23 scoring below the threshold. Visualization of the multiple alignment and LOGO calculation was performed using LaTex and the TexShade package v1.22 (Beitz, E. 2000). Specific conservation of residues in 4 different evolutionary groups (Metazoa, Chordata, Vertebrata, Mammalia) was evaluated using subfamily LOGO calculation (Beitz, E. 2006) with a significance threshold of 2 bits. Protein sequences were grouped accordingly to the taxonomic groups defined in Supplementary Table 1. The presence of N- or O-glycosylation sites on the human SIAE protein was predicted using NetNGlyc 1.0

S.Y., et al. 2013), while phosphorylation sites were predicted using NetPhos 2.0 (Blom, N., Gammeltoft, S., et al. 1999). The presence of N-terminal signal peptide was predicted using SignalP 4.1 (Petersen, T.N., Brunak, S., et al. 2011) and the overall probability of being a secreted protein was assessed using Secretome 2.0 (Bendtsen, J.D., Jensen, L.J., et al. 2004). In addition, the mouse NP_035864 sequence was used as a query in a BLAST search to retrieve 11 additional SIAE polypeptides from rodents.

Molecular modeling

Human SIAE protein sequence (NP_733746.1) was used in DELTA-BLAST against PDB database to retrieve suitable template structures for homology modeling. FASTA sequences of the three top hits (2APJ, 3PT5, 1ZMB) were aligned with human SIAE using MUSCLE and the alignment was manually refined to match the conservation blocks identified from the multi-species alignment. The refined alignment was then used as template in Modeller v.9.12 (Sali, A. and Blundell, T.L. 1993) to predict a structural model of human SIAE based on the crystal structure of 2APJ, 3PT5, 1ZMB, using two rounds of automatic loop refinement. Putative 3D structure of

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(http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 4.0 (Steentoft, C., Vakhrushev,

24 human SIAE was saved in pdb format and then visualized using PyMol v.1.7 (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC). Image of the topology of the secondary structure was generated using Pro-Origami (Stivala, A., Wybrow, M., et al. 2011) based on the pdb file. Multiple alignment with known protein structures from the three esterases 2APJ, 3PT5 and 1ZMB was used for the identification in human SIAE of the 4 sequence blocks characterizing family II SGNH hydrolases (esterase blocks) and of the residues involved in the catalytic process.

the predicted structure of human SIAE to reconstruct the hypothetical conformation of the catalytic crevice. To further support this region as the enzyme active site, we performed an automated molecular docking to identify the best interaction site between the human SIAE predicted structure and the enzyme substrate (9-O-acetyl-Neu5Ac). The structure of the enzyme substrate was generated in mol2 file format using Chemtool v.1.6.14 and then used in SwissDock (Grosdidier, A., Zoete, V., et al. 2011), together with the predicted structure of human SIAE. The automated blind docking was performed allowing a 3Å optimization of the input structures. The best predicted complexes (lower G value) were examined in PyMol to evaluate the interaction between the substrate, the 2 putative catalytic residues (S127 and H377) and the surface defined by the four-esterase blocks. We also selected as possibly involved in the active site all those residues placed within 5 Å from the substrate in the molecular model. Analysis of the surface defined by these residues allowed a better definition of the putative active site conformation.

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We analyzed the position of these 4 conserved blocks and the 4 putative catalytic residues in

25 Cloning and expression of SIAE in COS7 cells

A Mammalian Gene Collection cDNA clone corresponding to the SIAE lysosomal transcript (Lse) (MGC:87009; IMAGE:5278370) has been obtained from Geneservice Ltd. The coding region was amplified by PCR using cloned Pfu Turbo DNA polymerase (Stratagene). The resulting PCR product was then digested with the appropriate restriction enzymes and cloned into pMT21 vector (Kolodkin, A.L., Levengood, D.V., et al. 1997) to generate an ORF (Open

reverse (R) oligonucleotides were used in PCR amplification of SIAE ORF, HsSIAE F and HsSIAE R, bearing EcoRI and SalI restriction site, respectively, in order to obtain a directional cloning of the amplified product (Supplementary table 6). Amplification was carried out according to cycling parameters indicated for Pfu Turbo DNA polymerase for 35 cycles. Annealing step was performed at 55°C. The resulting recombinant vector (pMT21-Hs SIAE-Myc) was checked by automated DNA sequencing. pMT21-Hs SIAE-Myc construct was used to transfect COS7 cells (ATCC ® Number: CRL-1651™) kept in culture with Dulbecco's Modified Eagle's Medium (DMEM; Gibco-BRL) supplemented with 10% (v/v) FCS, 4 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For transient transfection, cells were seeded in 60 mm diameter Petri dish or on sterile glass coverslips (VWR International) in 24 well plates (Corning Life Science) and grown for 24 h before transfection. Transfection experiments were performed overnight in serum-free medium (OptiMEM, Gibco-BRL) using 2 g of plasmid DNA and FuGENE HD (Promega) reagent according to manufacturer’s guideline. Untransfected cells were used as negative controls. After 48 h expression, cells were washed in PBS (Gibco-BRL), collected by scraping, resuspended in sterile PBS (Gibco-BRL) supplemented with protease inhibitors (Protease Inhibitor Cocktail, Roche), and subjected to mild sonication (two 15” pulse at 4° C, 15%

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Reading Frame) encoding SIAE with a C-terminal myc-tag. The following forward (F) e

26 power, using a probe Sonicator Sonoplus-Bandelin electronic). The supernatant obtained after a centrifugation at 800 g for 10 min represented the crude cell extract and was used for protein quantitation, enzyme assay and Western-blot analysis.

Site directed mutagenesis Using the Quick-change II Site-directed Mutagenesis kit (Agilent) and specific mutagenesis primers (Supplementary table 6) we modified the 3 amino acids putatively involved in the

the enzyme, we generated 3 mutant constructs containing the following amino acid substitutions: T132A (pMT21-T132A), S332A (pMT21-S332A) and H377A (pMT21H377A). Each construct was cloned in XL-1 E. coli, purified with QIAfilter Plasmid Midi kit (Qiagen) and sequence verified. The resulting recombinant plasmids were used to transiently transfect COS7 cells as described in the previous paragraph.

Western-blot analysis

Media and cell extract samples were subjected to 12% (w/v) SDS-PAGE and subsequently transferred by electroblotting onto Hybond-P blotting membrane (GE Life Sciences). Membranes were blocked for 30 min in PBS containing 0.1% (v/v) Tween 20 (PBST) and 5% (w/v) dried milk and subsequently incubated for 1 h at 4°C with rabbit anti-Myc primary antibody (Millipore), diluted 1:500 in PBST containing 1% (w/v) dried milk. After several washes with PBST, membranes were incubated for 45 min at room temperature (RT) with donkey anti-rabbit HPR-conjugated secondary antibody (GE Life Sciences), diluted 1:5000 in PBST. After final washing in PBST, detection of immunocomplexes was carried out using SuperSignal West Pico Chemiluminescent Substrate detection kit (Thermo Scientific).

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enzyme active site. Starting from 50 ng of pMT21-SIAE representing the wild-type form of

27

Treatment with Tunicamycin, cycloheximide and PNGase-F

In vivo glycosylation was analyzed incubating COS7 cells with Tunicamycin (Sigma). Briefly, COS7 cells were transfected with pMT21-Hs SIAE-Myc and after 24 h expression tunicamycin (2 µg/ml) was added to the cell medium and cells were grown for further 24h. Cells and media were then collected and subjected to Western-blot analysis as described

just due to the over-expression, after 24h from the transfection we treated the COS7 cells with Cycloheximide (75 µM; Sigma) and let them grow for additional 24h. Any modifications in expression or glycosylation of SIAE protein were detected by Western-blot as previously described. Finally, to determinate if glycosylation is necessary for the activity of SIAE, cells extract were treated with PNGase-F (NEB) in native condition. Briefly, cells extracts corresponding to 20 µg were incubated over night at 37°C in G7 Reaction Buffer (0,5 M Sodium Phosphate pH 7,5; NEB) with or without PNGase-F. The enzyme activities of the samples were then analyzed as described below, while glycosylated and unglycosylated SAIE forms were detected by Western-blot. Untransfected cells were treated as above and used as control.

SIAE enzymatic assay

The enzymatic activity of SIAE in total cell lysates and in cell lysate and in media was determined using the general esterase artificial substrate 4-nitrophenyl acetate (pNPA) (Higa, H.H., Manzi, A., et al. 1989, Schauer, R., Reuter, G., et al. 1988). Briefly, pNPA dissolved in

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above. To clarify if the secretion of the protein was the normal fate of the SIAE or if it was

28 acetonitrile (80 mM) was added to 200 l of phosphate buffer (0,1 M) pH 8.4 to a 0,6 mM pNPA final concentration. For SIAE activity in cell extracts, 60 g proteins were used; for SIAE activity in growth media, 15 l were used as enzyme source. Release of 4-nitrophenol (pNP) was followed at 20°C at 405 nm using an ELISA plate reader (Scientific technology, Inc.). Enzyme activity was calculated using the extinction coefficient of pNP corresponding to 18.5 mM-1 cm-1 and expressed as nmol/min-1 (mU) mg-1 and mU ml-1 for cell extracts and media, respectively.

COS7 cells were grown on coverslips and transfected as described above. After 48 h cells were rinsed with ice-cold PBS, immediately fixed with 3% (w/v) paraformaldehyde in PBS for 15 min at RT and washed three times in PBS. Paraformaldehyde was quenched incubating samples with 50 mM NH4Cl in PBS for 15 min. After three PBS washes, cells were permeabilized with 0.3% saponin in PBS (PBS/Sap) for 30 min and then incubated with primary antibodies diluted in PBS/Sap for 1 h at RT. The following primary antibodies have been used: rabbit anti-Myc for SIAE (1:400, Millipore), mouse anti-GM130 for Golgi complex (1:300, BD), mouse anti-PDI for ER (1:400, Stressgen) and mouse anti-LAMP1 for lysosomes (1:300, BD). After washing three times in PBS/Sap, cells were incubated for 45 min at RT with donkey anti-rabbit Alexa 555 and donkey anti-mouse Alexa 488 secondary antibodies (1:400, Molecular Probes, Life Technology). Finally, after three washes with PBS/Sap followed by three washes with PBS, specimens were mounted using Fluorescent Mounting Mediun (Dako Cytomation), containing DAPI for nuclei labeling. Specimens were examined using a Confocal Laser System LSM 510 META (Zeiss) equipped with Axiovert 200 microscope (Zeiss). Image processing was performed with Adobe Photoshop software.

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Indirect immunofluorescence analysis

29

ACKNOLEDGEMENTS The financial support of MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) is acknowledged. This work was partly supported by Regione Lombardia and a Network Enable Drug Discovery (NEDD) grant to EM.

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39 LEGENDS TO FIGURES Figure 1 LOGO representation of multiple alignment of SIAE orthologous proteins The multiple sequence alignment of 32 sequences from SIAE orthologous proteins is represented in LOGO format. Peptide sequence and amino acid numbering refers to human SIAE (NP_733746.1). Black lines indicate the 11 highly conserved sequence blocks, while the red line indicates the four conserved blocks typical of family II SGNH hydrolases.

spirals, indicating α-helices. They are named accordingly to the topological representation in figure 3A. The other predicted features are also reported: †, glycosylation sites; ‡, phosphorylation sites; *, catalytic residues; +, residues potentially interacting with substrate.

Figure 2 Conservation of glycosylation and phosphorylation predicted sites The conservation of the predicted glycosylation and phosphorylation sites were assessed from the multiple alignments of SIAE orthologous proteins. Subfamily LOGO analysis performed as described in Methods suggested differential conservation occurring at 5 N-glycosylation (A) and 2 O-glysosylation sites (B). The most conserved predicted phosphorylation sites thorough Eukarya and Metazoa are also reported (C).

Figure 3 Predicted structure of human SIAE The structure of the human SIAE protein was predicted using homology modeling based on the known structures 2APJ, 3PT5 and 1ZMB. (A) Topological representation of the predicted structure comprise a core region, with 7 -sheets, depicted as orange arrows, and 9 -helices,

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Secondary structure elements are reported as black arrows, indicating β-sheets, or black

40 named A-I and depicted as marine blue cylinders. The C-terminal portion that poorly aligns with the template structures comprises 3-helices and is represented in blue. The core region of the structure, as seen in top (B) and lateral (C) cartoon representations, resembles the classical organization of family II SGNH hydrolases with 7 β-sheets buried within the αhelices. The 9-O-Acetyl-Neu5Ac molecule is represented as yellow sticks and placed as resulted from automated molecular docking, indicating the position of the putative active site.

Predicted active site of human SIAE Analysis of the predicted structure and molecular docking, allowed the identification of a putative catalytic site. The conformation of the catalytic surface (A) is suitable to accept the 9-O-Acetyl-NeuAc molecule, used as substrate, and resulted in a swallow pocket able to accept also complex glycans terminating with the acetylated NeuAc. The red portion of the surface is composed by the residues S127, T132 and H377 identified as the catalytic triad. Their placement (B) is compatible with the known mechanism of action on the O linkage at carbon 9. For the functional role played by T123 and H377, see also Figure 6.

Figure 5 Expression of SIAE in COS7 cells A) Rate of hydrolysis of 4-nitrophenyl-acetate in the range of pH from 3.0 to 9.0. Two different buffer systems were used: citrate buffer from pH 3.0 to pH 6.0 (black triangle) and phosphate buffer from pH 6.0 to pH 9.0 (black circle). B) Representative Western-blot analysis of crude cell extracts (20µg) and culture media (20µl) from non transfected cells (NT) and cells transiently expressing SIAE for 24h and 48h in absence or in presence of tunicamycin (Tun) or Cycloheximide (Cyc) treatment (24h + 24h). C) Left panel: Western-

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Figure 4

41 blot analysis of crude cell extracts (10µg) from NT cells and cells transiently expressing SIAE for 48h with or without Peptide-N-Glycosidase-F (PNGase-F) treatment. Right panel: SIAE activity measured in the same samples. For details see Material and Methods section.

Figure 6 Expression of SIAE mutants in COS7 cells Enzyme activity (upper panel) and representative Western-blot analysis (lower panel) of (A)

(NT), cells transiently expressing for 48h the wild-type SIAE (WT) and T132A, S332A and H377A mutants. Alanine substitution at position 132 and 377 resulted in a significant reduction in SIAE activity (p

Human sialic acid acetyl esterase: Towards a better understanding of a puzzling enzyme.

Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been o...
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