Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.


52
Sperm capacitation is a phenomenon first described by Chang, who demonstrated that freshly 53 ejaculated spermatozoa are unable to fertilize the oocyte immediately [1]. Rather, a period within 54 the female reproductive tract was required [1]. During this period, it is evident that spermatozoa 55 undergo a series of biochemical and metabolic changes to become fully capable of fertilizing the 56 egg [2][3][4]. From a metabolic perspective, Fraser and Lane first described the phenomenon of a 57 metabolic switch that occurs during capacitation [5]. In this context, freshly ejaculated may be triggered by the release of neuraminidases present at the sperm surface [27]. If this is the 88 case, then the presence of oviduct fluid components [21] heparin [22] and/or albumin [28] in the 89 medium probably facilitate this release [27]. 90 Despite the data that show changes in sperm sialylation content during capacitation, no single 91 publication has ever look at which proteins are sialylated in spermatozoa, yet alone quantified their 92 levels during capacitation. With this in mind, we used titanium dioxide (TiO2) followed by peptide- precipitated using methanol and chloroform [32]. Samples were incubated overnight (37 o C) with 131 trypsin at a 1:50 (trypsin/protein) ratio. Proteases were inactivated (bath sonication, 15 minutes) 132 and peptides were treated with alkaline phosphatase (20 U, 2 hours, 30 o C). 133 Enrichment of glycopeptides containing terminal Sia was performed as previously described [33].

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In brief, peptide samples were diluted in loading buffer [1 M glycolic acid, 80% (v/v)  Asn residue had to be flanked by the glycosylation consensus motif (NXS/T, where X is any amino 150 acid besides proline) which was manually validated. Peptides that were assigned a deamidation 151 event based solely on the MS data (i.e., no yor bfragment ion for a particular deamidated Asn 152 residue could be detected) were presumed to be glycosylated only if a canonical N-glycan motif 153 was present.

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The derived mass spectrometry datasets on the 3D-trap system were combined into protein 155 compilations using the ProteinExtractor functionality of Proteinscape 2.1.0 573 (Bruker Daltonics,156 Bremen, Germany). In order to exclude false positive identifications, peptides with Mascot scores 157 below 40 were rejected. Peptides with a mascot score above 40 were manually validated in 158 BioTools (Bruker Daltonics, Bremen, Germany) on a residue-by residue basis using the raw data 159 to ensure accuracy as previously described [33]   The phospholipase A1 (PLA1) activity of EL in intact mouse spermatozoa was analyzed using a 175 dye labeled-PLA1 specific substrate [35]. Sperm samples were divided into six groups; non-176 capacitated with or without H89 or A23187 and capacitated with or without H89 or A23187. The 177 compound H89 was supplemented 10 min prior to the addition of pentoxifylline and dbcAMP.

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After capacitation for 1h, ionophore A23187 was added to cells (20 μM final concentration) after 179 30 min of incubation and then samples were allowed to incubate for another 30 min. Spermatozoa 180 were washed (300 x g, 3 min) twice using BWW media with BSA and then once with BWW media 181 without BSA. Sperm pellets were then resuspended in BWW media without BSA to a final 182 concentration of 20 x 10 6 sperm/ mL.  HEK293T cells were used for transfection with ACO2-EGFP plasmid and ACO2-(HIS)6 plasmid 240 including the WT and N612D versions of these plasmids. We confirmed that human cells would 241 be suitable for transfection with the mouse ACO2 gene due to almost identical sequences. Cells 242 were split into 6-well plates to attain ~50% confluence the following day. Transfection was done 243 with 5 μg plasmid DNA and 10 μg PEI in 3 mL media. Firstly, 5 μg of plasmid and 10 μg PEI 244 were put into separate tubes and 150 μL DMEM (no FBS) added to each one. These were then 245 mixed together and incubated for 30 min. Next, media in the 6-well plates was replaced with 2.7 246 mL of fresh DMEM and the 300 μL DNA:PEI mix added. Cells were left to transfect for the times 247 indicated and then harvested. ACO2-GFP cells were fixed using 4% paraformaldehyde for 248 microscopy and FACS analysis or frozen at -80°C for immunoblotting. ACO2-(HIS)6 cells were 249 frozen at -80°C for immunoblotting or Aconitase assay. imidazole, 0.5% tween) and once in kit assay buffer. Afterwards, beads were resuspended in 100 261 μL kit assay buffer, 10 μL activation solution was added, and samples were rolled for 1 h at 4°C.

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One hundred μL of reaction mix was added to each tube and samples rolled at room temperature 263 overnight.

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Day 2: Frozen non-and capacitated sperm cells were thawed on ice. Cells were resuspended in 265 100 L kit assay buffer, sonicated and centrifuged (16,000 g; 4°C) for 15 min. Ten μL of activation 266 solution was added and samples incubated for 1 h on ice. Afterwards, 100 L reaction mix was 267 added to each tube and samples incubated at room temperature for 2.5 h. Standards were made 268 according to instructions and allowed to incubate for 30 min. The beads from day-1 preparation 269 were centrifuged and, together with the non-and capacitated sperm prepared on day 2, were loaded 270 in a 96-well plate in 100 μL duplicates. Ten μL of developer was added, samples incubated at 271 room temperature 25°C and read at 450 nm.

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Immunoblots of WT and mutant ACO2 were essentially performed as previously described [34].

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In order to determine the efficiency of the protocol used here to induce sperm capacitation, tyrosine The protocol to enrich for sialylated N-linked glycopeptides is shown in Figure 2 impairing their quantification in some biological replicates. These cases are indicated in Table 1 387 as not determined (ND) since we were not confident to report these further. Of interest, nine (6.3%) 388 of the glycopeptides identified here underwent significant changes during sperm capacitation.

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These glycopeptides belong to the proteins: ACO2, C4b-binding protein (C4BP), EL, inactive 390 serine protease 39 (PRSS39, also known as testicular-specific serine protease 1; TESP1), serine 391 protease 52 (PRSS52, also known as testicular-specific serine protease 3; TESP3), testis-expressed 392 protein 101 (TEX101) and zonadhesin (Table 1) Figure 3, the total amount and the molecular weight of EL did 408 not change after capacitation (Fig. 3A, lanes 1 and 2). In addition, the use of the PKA inhibitor 409 H89 during capacitation also did not affect the amount of EL (Fig. 3A, lanes 2 and 3). To 410 demonstrate equal loading, we re-probed the sample with anti-tubulin antibody (Fig. 3B). Using 411 the software Image J, the quantitative values of each band were plotted to confirm that no 412 significant change occur in EL expression after capacitation (Fig. 3C).

413
To verify whether the protein EL is redistributed in spermatozoa during capacitation, we 414 performed immunostaining using anti-EL antibody. Immunostaining for EL was observed in both 415 head and tail regions of non-capacitated (Fig. 4a,c) and capacitated (Fig. 4b,d)  staining. In addition, the staining intensity of the postacrosomal sheath showed high variation 418 among cells; being absent in some cases (Fig. 4b, white arrow exemplifies this variation). In the 419 tail region, although the midpiece and the cytoplasmic droplet showed high staining for EL, we 420 noted that some cells exhibited weaker labeling of the midpiece (Fig. 4c, non-and capacitated spermatozoa were induced to undergo the acrosome reaction. These sperm 434 cells were subsequently washed, then the level of intact, partial or complete acrosome loss were 435 measured using FITC-PNA staining. As shown in Figure 5, capacitated sperm plus ionophore 436 A23187 had the highest level of complete acrosomal loss as expected.

437
To determine whether a change in EL activity occurred during capacitation, we measured PLA1 438 activity before and after capacitation, together with the inhibitor H89 or the acrosomal-inducer, 439 ionophore A23187. We observed a significant loss in the PLA1 activity of EL during capacitation Spermatozoa are catabolic in nature and, as such, it is not a surprise that the majority of Sia residues 452 are lost during capacitation. However, in this study, we observed one enzyme that had an increase 453 in sialylation, namely ACO2. To further understand this finding, we measured total Aconitase 454 activity before and following mouse capacitation. As shown, during capacitation a statistically 455 significant decrease in the level of Aconitase activity was observed (Fig. 7). Although this 456 measurement would include both cytoplasmic and mitochondrial Aconitase activity, we reasoned 457 that sperm have very little cytoplasm, therefore, the bulk of Aconitase activity should be from the 458 mitochondrial form.

459
To confirm that a decrease in ACO2 activity occurs specifically through sialylation at N612, we 460 made both WT and Sia mimic, whereby the N612 was replaced by the negatively charged Aspartic 461 acid (N612D). In both cases (WT and mimic), we made a GFP-and a HIS-tagged separate proteins.

476
We next measured the ACO2 activity of the WT and N612E mutant (Fig. 10) N614 (Asn615) and Gln563 which is present on an adjacent loop (Fig 11A,B). This data suggest 494 that inhibition of Aconitase activity, through siltation of Aspargine 612, is due to major distortion 495 of the active site which prevents catalysis from occurring.

498
Despite the importance of capacitation, the molecular mechanisms underlying this process are not 499 yet fully understood. Previous studies have suggested that one facet of capacitation is a loss in Sia 500 residues, which may be modulated by one (humans) or two (mouse) neuraminidases, namely 501 neuraminidase 1 and 3 [27]. In the present study, using a LC-MS/MS-based approach, we were 502 able to investigate capacitation-related changes of N-linked glycoproteins bearing terminal Sia.
503 Surprisingly, we found very little regulation of Sia within proteins groups following in vitro 504 capacitation, with only 6.3% (9 of 142 peptides) demonstrating a significant change.

505
According to our data, the enzyme EL is one of the sperm proteins in which the Sia content is 506 altered during capacitation. Previous studies have shown that four (N62, N118, N375, and N473) 507 of the five potential N-glycosylation sites of human EL are occupied by glycan moieties [51,52].

508
In the present study, the sugar moiety at the N62 glycosylation site of EL was found to contain Sia 509 in its structure. In addition, the peptide containing this glycosylation site was significantly reduced 510 after in vitro capacitation, despite the fact that both quantity and molecular weight of EL remained 511 unchanged. This suggests that a loss of a small glycan moiety or of a Sia residue itself occurs 512 within EL during capacitation. Notably, a sialylated N-glycan structure at the corresponding 513 glycosylation site (N64) has also been identified by our group in the EL of rat sperm [33]. In this 514 case, spermatozoa were taken from the caput, corpus and cauda regions of the epididymis.

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Remarkably, the Sia residue within EL was only found in spermatozoa derived from the caudal 516 location. Furthermore, we have observed that the amount of EL within rat spermatozoa does not 517 change, suggesting that Sia is added to EL during epididymal transit (data not published; [33]).

518
Given that Sia residues are removed from the same glycosylation site during capacitation, it is 519 likely that this glycan moiety plays a specific role in regulating EL enzyme activity.

520
The glycosylation site at N62 of mouse EL is a conserved feature among animals and other 521 members of the triglyceride lipase gene family, such as lipoprotein lipase and hepatic lipase. Using 522 recombinant proteins, two separate studies have produced point mutations of the amino acids that 523 are glycosylated in EL. Interestingly, in both cases, the loss of N62 led to increased EL activity 524 [51,52]. Due to their negative charge and hydrophilicity, Sia residues within this glycan moiety 525 could influence the structure and/or substrate specificity of EL, therefore, regulating its enzymatic 526 activity. Of note however, we observed a decrease in the PLA1 activity of EL following reduction 527 of its sialylated glycopeptides (N62 glycosylation site) during capacitation. We can only assume 528 that, besides the loss of N62, EL is likely to be regulated in other (as yet unknown) ways in order 529 to switch off its activity.

530
In addition to EL, we found N612 sialytion of ACO2 increase following in vitro capacitation. This 531 enzyme catalyzes the non-redox reaction of the TCA cycle in which stereo-specific isomerization 532 of citrate to isocitrate occurs [53]. Adequate supply of ATP is essential to support capacitation-533 associated changes such as hyperactivation [54]. In mouse, it is fairly well understood that, during 534 capacitation, there is a switch from oxidative phosphorylation over to glycolysis [5]. Thus, non-535 capacitated sperm show high oxygen consumption, which diminishes as sperm make the switch 536 over to glycolysis during capacitation [5]. Herein, such a switch could be brought about through 537 sialylation of ACO2 particularly N612. Indeed, modelling of the enzyme suggests that the Asn 538 group sits atop of the ACO2 activity site (Fig. 11)  Inspection of Uniprot suggests that the glycosylation at N612 has never been reported in 555 any other cell type and, as such, may represent a novel mechanism, attributed just to sperm cells.

556
Unfortunately, compounds to block or inhibit sialic acid transferases are not cell permeable and, 557 for this reason, we were unable to directly ascribe the significance of a lack of ACO2 activity to  Aconitase activity was measured. To ensure equal amounts of Aconitase were present, the lysate 757 was precipitated, run into SDS page, transferred and probed using anti-Aconitase antibody. The 758 data shown is the average (± SD) of four biological replicates. Asterisk represents statistical 759 significance (p < 0.01).