Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis

Kunio Kawanishi, … , Anja Münster-Kühnel, Ajit Varki

J Clin Invest. 2020. https://doi.org/10.1172/JCI137681.

Research In-Press Preview Metabolism Nephrology

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Find the latest version: https://jci.me/137681/pdf Evolutionary Conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis

Kunio Kawanishi1,2#*, Sudeshna Saha1,2*, Sandra Diaz1,2, Michael Vaill1,2,3, Aniruddha Sasmal1,2, Shoib S. Siddiqui1,2##, Biswa Choudhury1, Kumar Sharma4###, Xi Chen5, Ian C. Schoenhofen6, Chihiro Sato7, Ken Kitajima7, Hudson H. Freeze8, Anja Münster-Kühnel9, and Ajit Varki1, 2, 3, 4**. 1Glycobiology Research and Training Center, 2Department of Cellular & Molecular Medicine, 3Center for Academic Research and Training in Anthropogeny, and 4Department of Medicine, University of California, San Diego, La Jolla, CA, USA. 5Department of Chemistry, University of California, Davis, Davis, CA, USA 6Human Health Therapeutics Research Center, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada, 7Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan. 8Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA. 9 Clinical Biochemistry, Hannover Medical School, Hannover, Germany. * The contributions of the first two authors are considered equal. #Current Address: Kidney and Vascular Pathology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan ##Current Address: American University of Ras Al Khaimah (AURAK), United Arab Emirates (UAE) ###Current Address: Center for Renal Precision Medicine, Division of Nephrology, Department of Medicine, University of Texas Health San Antonio **Correspondence: [email protected], University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA.

Conflict of interest: The authors have declared that no conflict of interest exist. Abstract (200 Words) Human metabolic incorporation of non-human sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via pre-existing antibodies, likely contributing to red- meat-induced atherosclerosis acceleration. Exploring if this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-D- glycero-D-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. Levels of Kdn precursor mannose also increased but within normal range in ESRD patients. Mannose ingestion by healthy volunteers raised urinary mannose and Kdn. Kdn production pathways remain conserved in mammals but were diminished by a M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in two lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that potential toxicities of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals likely conserved Kdn biosynthesis and modulated it in lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates/metabolites. However, human cells can be forced to express Kdn-glycans, via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing CMP-Kdn. Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Keywords: 2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (Kdn), mannose, end-stage renal disease (ESRD), UDP-N-acetylglucosamine 2-epimerase (GNE), phosphomannose isomerase (PMI), N-acetylneuraminate synthase (NANS), N-acylneuraminate cytidylyltransferase (CMAS)

2 Introduction Sialic acids (Sias) are a diverse family of nine-carbon backbone monosaccharides found in glycosidic linkage to outer tips of the glycan forest that covers all vertebrate cells and most secreted macromolecules (1–5). Given their widespread expression and localization, Sias have diverse roles in biology, evolution and disease (2, 6–10). The two most common mammalian Sias are N- acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is hydroxylated from Neu5Ac by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). Humans cannot synthesize Neu5Gc due to inactivation of the CMAH gene about 2-3 million years ago (11). We previously reported that most humans have circulating polyclonal antibodies against glycans bearing Neu5Gc (12, 13) and that anti-Neu5Gc-glycan antibody generation correlates with introduction of Neu5Gc during weaning (14). When exogenous Neu5Gc of dietary origin gets metabolically incorporated at sites such as epithelia and endothelia (15), glycans bearing this Sia act as foreign “xeno-autoantigens”, triggering inflammatory responses termed “xenosialitis” (13, 16–19). Disease-escalating effects of xenosialitis have been demonstrated in cancer and atherosclerosis models using human-like Cmah null mice (20, 21).

2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) is another vertebrate Sia first discovered in the cortical alveolar polysialoglycoprotein of rainbow trout eggs (22), and is abundant in cold-blooded vertebrates. While glycosidically-conjugated Kdn was originally not found in mammals (23), free Kdn was detected in porcine submaxillary glands (23), cow milk products, human urine (24), and porcine milk (25). Elevated levels of free Kdn (and indirect evidence suggesting very low levels of conjugated Kdn) have also been reported in human ovarian (26) and throat cancers (27), and free cytosolic Kdn-containing N-glycans accumulate in human prostate cancers (28). Monoclonal anti-Kdn antibodies produced from Kdn-immunized mice also detected very small amounts of Kdn in rat pancreas (29, 30). Another monoclonal antibody against poly (2-8-linked) Kdn indicated the presence of this structure on megalin in the kidney (31–33), on certain cancer types (33), as well as on ceruloplasmin (34). However, to our knowledge, conclusive evidence for occurrence of Kdn-glycans in normal human tissues are still missing. Free Kdn is synthesized in the cytosol (35, 36) (Figure 1) from mannose 6-phosphate (Man- 6P) (C-2 epimer of glucose 6-phosphate), which is itself used for biosynthesis of N-linked glycans found on most secretory proteins, as a core component of GPI-anchors, and as a metabolic precursor to fucose (37). Importantly, unlike many other free monosaccharides, mannose is very

3 efficiently taken up by different cell types using a transporter that is insensitive to glucose (38, 39). Although specific mannose transporter(s) have not been identified yet, mannose uptake suggests that endogenous mannose production may sometimes be inadequate for cellular requirements, and that salvage of extracellular free mannose can be important. Oral mannose administration can directly contribute to N-glycan synthesis (40), however, Man-6P is also endogenously derived from fructose-6-phosphate (Fru-6P) through phosphomannose isomerase (PMI) (39) (Figure 1). Inherited deficiency of PMI results in congenital disorder of glycosylation CDG)-Ib (PMI-CDG) associated with protein loss in intestines, liver disease, hypoglycemia, and blood clotting disorders (41), and these patients respond well to oral mannose therapy (42). On the other hand, excessive mannose is toxic for honeybees (43, 44) and can blind baby PMI KO mice (45). In both instances there is a low ratio of PMI relative to hexokinase. This imbalance produces a metabolic flux in which mannose is rapidly phosphorylated, but Man-6P cannot efficiently be converted into glycolytic intermediates. Thus, ATP is wasted to produce a molecule without metabolic value to the cell, an effect that has been termed “Honeybee Syndrome” (44, 46). While oral mannose is a potential therapeutic nutraceutical to treat certain types of CDG type I disorders (47), mannose supplements are also used to suppress urinary tract infections by E. coli that bind mannose-rich glycans (48–50). Furthermore, recent murine studies showed that mannose supplementation have an immunosuppressive effect by inducing T regulatory cells (51) and ameliorate high fat induced obesity and glucose tolerance by changing the microbiome profile (52). Several human studies examined the importance of balanced plasma-mannose level in diabetes, in particular, insulin resistance (53, 54), obesity and cardiovascular disease risk (55), as well as colorectal cancer risk (56). A recent study also suggested that mannose administration impairs tumor growth and enhances chemotherapy (57). However, to date, no comprehensive study has been performed to understand potential mannose toxicity in humans or the possibility of regulation of physiological mannose homeostasis via conversion into Kdn. Here we report that while the serum free mannose levels are increased, but still maintained in an almost normal range in end-stage renal disease (ESRD) patients receiving hemodialysis, their free serum Kdn levels are significantly elevated. Furthermore, we show that humans not only excrete excess mannose into urine, but also metabolize it into free Kdn and excrete the Sia into urine presumably for modulating mannose levels.

4 We also assess evolutionary conservation (and/or convergent evolution) of genes encoding Kdn metabolic enzymes and hypothesize that the conservation of Kdn biosynthesis helps to detoxify excess Man-6P/Fru-6P. We also show that mannose-fed mammalian cells produce free Kdn in their cytosol with predicted increases in certain genetic mutants, and that humans can have antibodies against glycosidically-linked Kdn glycoconjugates (hereafter called Kdn-glycans).

Results

Increased circulating free Kdn in end-stage renal disease (ESRD) patients. Previous studies in human-like Cmah null mice showed that orally ingested glycosidically-conjugated Neu5Gc can be metabolically incorporated into tissues, most prominently into epithelial and endothelial cells, driving antibody- and complement-mediated inflammatory progression of carcinomas (20) and atherosclerosis (21). In contrast, ingested free Neu5Gc is excreted rapidly into the urine of both rodents (58, 59) and humans (15). On the other hand, sustained high-level exposure of cultured human cells to free Neu5Gc can result in incorporation, sufficient to generate human anti-Neu5Gc- glycan-antibody- and complement-mediated inflammation (60). Thus, we hypothesized that accumulation of free Neu5Gc from the diet contributes to accelerated atherosclerosis in ESRD patients. To explore this hypothesis, we examined free Sia profiles of sera collected from anonymized hemodialysis patients just prior to their weekly dialysis and compared them to samples from healthy volunteers. We did not see free Neu5Gc peaks in either ESRD or control sera, instead observed a significant accumulation of other DMB-reactive α-ketoacids including Kdn and Neu5Ac in ESRD samples (Figure 2, A-D, Supplemental Figure 1). Identity of Kdn and Neu5Ac peaks was confirmed by LC/MS (Supplemental Figure 2) using commercially available standards (Supplemental Table 1). Interestingly, we also noted a slight increase of 3-deoxy-D- manno-oct-2-ulosonic acid (Kdo, a bacterial octulosonic acid and component of plant polysaccharides) and other unknown peaks only in ESRD samples (Supplemental Figure 2). Serum free Kdn levels in ESRD patients are significantly elevated in comparison to healthy volunteer (2.91 (0.68) µM vs. 0.50 (0.25) µM, mean (SD), p<0.001) (Figure 2B), as well as serum free Neu5Ac (16.9 (2.01) µM vs. 2.34 (0.54) µM, p<0.001) (Figure 2C). Although levels of mannose (the metabolic precursor of Kdn) in ESRD were significantly higher than in healthy volunteers

5 (83.8 (15.1) µM vs. 44.5 (19.1) µM, p<0.001) (Figure 2D), the level in most ESRD individuals (ten of sixteen individuals) was within the normal range in humans (40-80 µM) (61).

Ingested mannose is partially converted into Kdn and excreted into urine in humans. We next asked if mannose feeding to healthy humans would result in increased Kdn production. In prior studies on normal humans feeding 0.10-0.25 g/kg body weight of mannose gave a peak concentration of mannose in blood of ~0.5 mM with only mild gastrointestinal symptoms in one individual. However, at oral doses exceeding 0.25g mannose/kg body weight approximately half of subjects reported mild gastrointestinal distress. No other symptoms were reported (47). We therefore studied healthy humans during mannose ingestion of 0.20 g/kg body weight. No obvious symptoms were reported, and serum mannose levels, measured by GS-MS, went up 1 hour post ingestion to a maximum of 285 µM (mean) and decreased subsequently within 8 hours post ingestion to 120 µM (Figure 3A) as previously described (47). Urine mannose levels increased from 4 µM (mean) (before mannose ingestion) to 11 µM and then decreased to about 5 µM (Figure 3A). Similarly, a peak of free Kdn was observed at 4 hours after ingestion and further kept at higher level (Figure 3B). Overall, following the serum mannose peak at 1 hour both mannose and free Kdn levels in urine went up within 4 hours post ingestion (Figure 3), suggesting that excess free mannose could be directly and indirectly (metabolized to free Kdn) eliminated by urinary secretion. Serum Neu5Ac and Kdn levels did not change after oral mannose administration, consistent with previous animal study (36) and our findings of serum Kdn elevation only in ESRD and not in control healthy individuals (Figure 2). To test whether oral mannose administration results in Man-6P accumulation in human serum and urine, we measured Man-6P concentration post 1 hour in the serum and post 4 hours in the urine of healthy humans. We could not detect any Man-6P using HPAEC-PAD, even after mannose ingestion (Supplemental Figure 3). One possible explanation could be that Man-6P is not exported out of the cells. Another possibility is the presence of plasma/serum phosphatases which destroy Man-6P, and that those enzymes do not exist in the urine. The latter possibility is supported by the observation that exogenously added Man-6P can only be detected in urine but not in serum samples (Supplemental Figure 3C, F). Overall, these results suggest that mannose level is possibly controlled either directly by urinary system and/or indirectly by Kdn metabolic pathways.

6 Dose-dependent mannose toxicity in cultured human and mouse cells. No report exists to confirm mannose toxicity in humans, so far. Free Kdn is synthesized in the cytosol of mammalian cells fed with 20 mM mannose (35, 36). However, in vitro and in vivo studies suggest that mannose toxicity in PMI KO cells or embryos can be caused by Man-6P accumulation, which inhibits glucose metabolism and depletes intracellular ATP (39, 40, 46). To test mannose toxicity in human cells, we used a continuous cell viability assay for mannose-fed cultures at varying concentration (0-200 mM), using another monosaccharide N-acetylglucosamine (GlcNAc) (which is not efficiently taken up) as a control for any hyperosmolar effect (Figure 4). Human embryonic kidney cells 293A (HEK293A) and human umbilical vein endothelial cells (HUVECs) were tested as representative cells of kidney epithelium and endothelium encountering mannose in blood stream, respectively. We also studied a human Burkitt's lymphoma line (BJAB K88, GNE+/+, GNE WT) and a subclone deficient in Sia production (BJAB K20, GNE-/-, GNE KO) (62), as well as PMI+/+ (PMI WT) and PMI-/- (PMI KO) mouse embryonic fibroblast cells (40). HEK293A (Figure 4A, B) and HUVEC (Figure 4C, D) did not show significant differences between mannose and GlcNAc, even at high concentration (100 mM). However, the other cell lines clearly showed selective mannose toxicity at a lower concentration (from 25 mM) (Figure 4E, G, I) in comparison to GlcNAc feeding (Figure 4F, H, J). Particularly the PMI KO cells when cultured at mannose concentrations >1 mM (Figure 4K), displayed the “Honeybee Syndrome”, caused by Man-6P overproduction as previously shown (39, 40). Previous study confirmed that PMI KO cells showed Man-6P accumulation in the physiological condition (20-100 µM mannose), and ATP depletion started when maximum levels of Man-6P had been reached (4-8 hours of culture in presence of 500 µM mannose supplementation) (40). We also observed reduction in the viability of PMI KO cells fed with GlcNAc (10-30 mM) (Figure 4L) by an yet unknown mechanism (63), and could be partly associated with ATP depletion during GlcNAc feedings. Notably, unlike all other cells tested for the effect of GlcNAc, PMI KO cells in Figure 4L were always maintained in mannose (25 µM) supplemented media, since this minimal mannose is indispensable for the viability of the PMI KO cells. Next, we wanted to determine if mannose feeding of the cultured cells could result in the production of cytosolic Kdn or if the Sia upon formation is excreted outside into the growth media. To that end, we grew different human and mouse cells including HEK293A, HUVEC, BJAB K88 (GNE WT) and K20 (GNE KO) as well as PMI WT and PMI KO mouse embryonic fibroblast

7 cells in presence of exogenous free mannose. Based on the previous Kdn studies with 20 mM mannose supplementation (35, 36), and our viability assay (Figure 4), we first tested 0, 1, 3, 5, 10 and 15 mM mannose supplementation and analyzed the concentration of Kdn relative to Neu5Ac in the cytosol (Supplemental Table 2-1) or in the growth media (Supplemental Table 2-2) by DMB- HPLC analysis. We decided to use 0 and 15 mM mannose supplemented conditions for all the human and PMI WT cells for further quantification (n=3 experiments) (Figure 5). Since PMI KO cells fail to survive in absence of mannose and require a minimal mannose concentration for their viability during the assay, we could not quantify Kdn in the mannose-depleted media for these cells. Instead, we used 25 µM (minimal concentration for cell survival), 250 µM (mimicking the maximum concentration in human sera, Figure 3A) and 1 mM mannose supplementation for the Sia quantification in PMI KO cells (n=3 experiments) (Figure 5). Analyses of purified free Sia and CMP-Sia by DMB-HPLC analyses confirmed that only CMP-Sia survives reduction by NaBH4 (64) (Supplemental Figure 4), allowing one to distinguish between free Sia and CMP-Sia in the cytosol. Most of the cytosolic Kdn represented the free form (NaBH4 sensitive) (Figure 5 A-E) (up to 1000 pmole Kdn /million cells by DMB-HPLC) and not CMP-Kdn (NaBH4 resistant) (Figure 5 F-J) (less than 60 pmole Kdn /million cells). Unlike the other human cell lines which showed an increase in the cytosolic accumulation of Kdn, cytosolic free (and CMP-) Kdn levels in HEK293A cells did not change upon mannose feeding (Figure 5A). Instead, the levels of Kdn in the media of HEK293A were found to be elevated (Supplemental Table 2-2). We also analyzed an immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2) (65), which showed significant elevation of Kdn in media after mannose feeding (Supplemental Table 2-2). Taken together with Sia analyses in cytosol and media, we conclude that human and mouse cells can produce free Kdn, and secrete it from cells, by an as yet unknown mechanism to possibly modulate excess Man-6P.

Glycosidically-conjugated Kdn rarely occurs in mammalian glycans. For a better understanding of the Kdn sialoglycome in eukaryotes, we searched available online databases for glycans with terminal glycosidically-conjugated Kdn. While glycosidically-conjugated Neu5Ac and Neu5Gc were commonly observed in different eukaryotic groups, such Kdn-glycans were only reported in fish and amphibians, and not in any mammalian cells (Table 1). Indeed, in an extensive and in-depth mass-spectrometric study of N-glycans from murine and human cell lines and tissues

8 conducted by the Consortium for Functional Glycomics (66) (http://www.functionalglycomics.org) there was no evidence for Kdn-glycans in approximately 425 human and mouse cell and tissue samples studied (Anne Dell and Stuart Haslam, Imperial College London, London; personal communication). Taken together these data suggest that the capacity for utilizing Kdn as a Sia conjugated to glycans was diminished or eliminated prior to the time that vertebrates became primarily land-based.

The Kdn biosynthetic pathway is conserved throughout vertebrate phylogeny and is under purifying selection in humans. As mentioned above, there is currently no conclusive evidence of Kdn-glycans in healthy mammalian tissues. Based on our observation of free Kdn in urine following mannose feeding (Figure 3B), we therefore asked if human cells contain Kdn biosynthetic enzymes required for the production of Kdn from Man-6P. We investigated seven human genes involved in mannose metabolism and conversion of Man-6P to Kdn (Figure 1) for evolutionary conservation, comparing them to respective homologous genes in five species representatives of vertebrate phylogeny (Table 2). While previous studies identified conservation of Sia biosynthesis pathways in mammals (67), our results demonstrate that the enzymes capable of synthesizing Kdn from Man-6P are also conserved throughout vertebrate phylogeny and are experiencing ongoing purifying selection in humans and other vertebrate species [Table 2 0.005 – 0.347 Ka/Ks ratios, 64 - 89 % nucleotide (nt), and 54 - 99 % amino acid (AA) conservation]. This finding further raises the question as to why Kdn biosynthesis is under purifying selection in organisms that do not appear to possess the capacity to utilize the monosaccharide in the synthesis of glycan structures. One explanation is that most of these genes are also involved in other conserved metabolic pathways. However, another not mutually exclusive explanation of this selective pressure (i.e. preserving Kdn production) would be the necessity of these species to avoid toxicity of excess Man-6P (see below).

Single amino acid in sialic acid 9-phosphate synthase that alters the rate of mannose to Kdn conversion is restored independently in two mammalian lineages, including humans. An earlier study showed that a single amino acid (AA) exchange [methionine 42 (M42) to any other AA except leucine] abolishes the ability of human sialic acid 9-phosphate synthase (NANS) to synthesize Kdn-9P while retaining Neu5Ac-9P biosynthetic ability in vitro (68). We

9 investigated the conservation of this key M42 position in other vertebrates. NANS protein sequences were highly conserved in a wide variety of lineages, e.g. mammalian species showed 87-100% Identity (I) and 94-100% Homology (H), while non-mammalian vertebrates showed not less than 76% I and 89% H (Supplemental Table 3). Phylogenetic topology including predicted ancestral AA and respective codons corresponding to M42 in human NANS was studied (Figure 6, Supplementary Figure 5). Considering the high homology to the human enzyme, we hypothesized that high Kdn-9P synthase activity was presumably associated with M42, also present in common ancestor. Remarkably, M42 was mainly found in non-mammalian vertebrates, e.g. Aves, Amphibia and Actinopterygii, for the latter two of which the presence of glycosidically- conjugated Kdn has been unequivocally demonstrated by MS analyses (Table 1). In mammals, M42 was found in Monotremes (e.g. Platypus) and Metatheria (e.g. Koala and Opossum). In Placentals, M42 was initially replaced by less active T42 but subsequently restored independently in two lineages, Primates and Artiodactyla (even-toed ungulates and cetaceans), apparently by convergent evolution. This finding again suggests on-going selection possibly for the need to modulate excess Man-6P into Kdn in clades that no longer use Kdn as a glycosidically-conjugated Sia on glycoconjugates.

Mannose feeding in mutant cells can cause Kdn incorporation onto cell surfaces. Since healthy mammalian cells do not present glycosidically-conjugated Kdn, we decided to include mutant cell lines in the analyses. Based on the cell viability assay results shown above, the highest concentration of mannose in cell culture media was determined to be 15 mM (1 mM for PMI KO cells) to maintain optimal cell viability. As mentioned earlier, the total amount of free (and CMP- ) Kdn in cytosol or media were elevated in all types of cells after mannose supplementation (Figure 5, Supplemental Table 2), but no significant increase in CMP-Kdn concentration (Figure 5). To evaluate the possible production of Kdn-containing glycan structures, we analyzed the

Sia content of various cell lines fed with mannose, treated with NaOH and NaBH4 to destroy any unconjugated Sia and then examined for glycosidically-conjugated Sias from the total cell lysate by DMB-HPLC (Supplemental Table 2-3). The highest Kdn/Neu5Ac ratio was observed in BJAB K20 cells, which in consequence of a GNE mutation cannot make internal Neu5Ac. In line with this, BJAB K20 also show a high amount of free Kdn in the cytosol as well as in the media (Supplemental Table 2-1, 2-2). However, total conjugated Kdn were much lower than free and

10 CMP- Kdn levels in cytosol and media even in BJAB K20 (Supplemental Table 2-1, 2-2, 2-3). We also collected membrane fractions after ultracentrifugation and DMB-HPLC analyses showed conjugated Kdn in the membrane fraction, especially in BJAB K20 and PMI KO (Supplemental Table 2-4). Next we used two previously reported anti-Kdn-glycan monoclonal antibodies, kdn3G (69) and kdn8kdn (70), which have specific affinities to glycans containing α2-3- or α2-8-linked Kdn, respectively, but not α2-6-Kdn glycan-structures (29, 69). Flow cytometry using these anti- Kdn antibodies did not detect any peak shift for BJAB K20 cells (Figure 7A, B), probably because BJAB K20 cells mainly form α2-6-linked Sia which are undetectable with the two antibodies used for the assay (71, 72). PMI KO cells present small amounts of Kdn in α2-3- and α2-8-linkage on cell surface structures upon feeding with mannose (Figure 7C, D) in contrast to BJAB K20.

Fish Cmas gene expression plus mannose feeding can force Kdn incorporation onto some normal cell surfaces. Prior to transfer to glycans, Sias are activated into CMP-Sias, a reaction involving CTP and catalyzed by CMP-Sia synthase, encoded by the CMAS gene. As we showed earlier, Kdn incorporation into cell surface glycans seems to be rare in comparison to free Kdn production even after mannose feeding, likely because human CMAS and mouse CMAS, preferentially work on Neu5Ac (73–75). Indeed, human CMAS has 94% identity to the murine enzyme (75, 76) (Supplemental Table 3), and the activity of the recombinant murine CMAS was 15 times lower for Kdn compared to Neu5Ac (75). Our investigation into the phylogenetics of these pathways brought attention to a whole genome duplication event that occurred in the teleost- lineage of fish (77). This duplication produced two fish Cmas paralogues known as Cmas1 and Cmas2 (78). Since our previous results showed HEK293A cells mainly make and excrete excess free Kdn into media, recombinant zebrafish Cmas plasmid vectors were transfected into HEK293A (Figure 7E) (30-40% of cells are transfected, by checking transfection efficiency by using anti- Flag antibody, data not shown). Flow cytometry using anti-Kdn antibodies did not show any significant peak shift in Cmas transfected HEK293A cells fed with up to 15 mM mannose (data not shown). However, DMB-HPLC analysis of the membrane fraction showed that zebrafish Cmas transfection can result in glycosidically-conjugated Kdn (exemplarily shown for Cmas2 transfected HEK293A cells in Figure 7H, I). The ratio of glycosidically-conjugated Kdn/Neu5Ac, especially in mannose fed samples, was lower in Cmas1 transfected cells (Supplemental table 2- 5), consistent with differential substrate specificity in vitro (78). Interestingly, the amount of

11 conjugated Neu5Gc (traces derived from fetal calf serum) is lower than Neu5Ac but still higher than Kdn in human cells (Figure 7F-I), confirming a limited utility for Kdn compared to Neu5Gc in glycosylation.

Humans have circulating polyclonal antibodies against Kdn glycans. We have previously shown that humans have varying levels of circulating antibodies against glycans bearing the non- human Sia Neu5Gc (13, 14, 79). While glycosidically-conjugated Kdn is not reported in normal human tissues, some malignant cells may have the potential to incorporate Kdn on their surface sialoglycans in a Kdn-rich environment. To probe for the presence of any circulating anti-Kdn- glycan antibodies, we used chemoenzymatically synthesized biotinylated glycan containing Kdn α2-3-linked to Galβ1-4GlcNAc (Figure 8A) to screen commercially available pooled normal human sera in an ELISA based assay and found the presence of IgG antibodies against this Kdn- glycan epitope (Figure 8B). We also used Neu5Ac and Neu5Gc linked sialoglycans alongside nonsialylated glycans containing the same underlying Galβ1-4GlcNAc epitopes to determine the presence of antibodies against those corresponding epitopes (Figure 8A, B). While our current data do not present the complete repertoire of anti-Kdn-glycan antibodies in human, it provides evidence for the selective occurrence of such antibodies in normal human sera.

Discussion Mannose levels in mammalian sera are variable but seem to be controlled in a restricted range (20-160 µM) (61). Levels can be affected by ingestion of mannose-rich food as fruits, vegetables, yeasts, etc.(46, 80), lysosomal release by N-glycan processing (61, 81), and recovery and reabsorption by sodium glucose cotransporters (SGLTs) (82). SGLTs may work as mannose transporters in both intestine and kidney (82, 83). Mannose is also transported into mammalian cells via glucose transporters, but mannose-specific transporters have not yet been identified (84). Almost all transported mannose (95-98%) is catabolized via PMI, and in [2-3H]-mannose radioactive metabolic labeling experiments only up to 2% of mannose taken up is used for N- glycosylation (61, 81). Interestingly a later study using more sensitive 13C-glucose and 2H- mannose isotopic labeling and GC-MS analysis showed that transported mannose is utilized more

12 efficiently than glucose: while 1.8% of transported mannose appears in N-glycans, only 0.026% of transported glucose is utilized in N-glycan synthesis (85). Furthermore, mannose from lysosomal N-glycan processing is transported outside the cell as free mannose without being catabolized, through a nocodazole-sensitive transporter (81). The reason for these separate pathways for intracellular mannose is unknown, but cells may need to eliminate excess mannose to avoid accumulation of toxic products such as Man-6P. Indeed, mannose toxicity was confirmed in our cell viability assay most prominently in PMI KO cells (Figure 4). High levels of mannose are also teratogenic in rat-embryo cultures and pregnancy models, due to inhibition of glycolysis (86, 87). Free Man-6P might directly be metabolized to Kdn or through the hexosamine biosynthetic pathway (Figure 1). While human cells can synthesize Kdn from Man-6P, Kdn is not (or only in very small quantities) incorporated in glycoconjugates in healthy humans. Given all these observations, we hypothesize that Kdn synthesis is conserved in mammalian metabolism to buffer cellular levels of Man-6P. Concordantly, our phylogenetic sequence analysis suggests an undergoing evolutionary pressure to conserve the metabolic pathway that converts mannose to Kdn in vertebrates (Table 2). However, the capability to incorporate Kdn into glycoconjugates differs between vertebrate classes. The apparent lack of Kdn in the glycan determinants of mammals contrasts strikingly with the previously mentioned examples of Kdn-containing structures in the gametes and mucus secretions of amphibia and fishes and suggests that the enzymes necessary for Sia incorporation may have lost their activity towards Kdn since the relatively ancient divergence of warm- and cold-blooded vertebrates. For a free monosaccharide to be incorporated into glycan structure it must first be activated to a high-energy donor, e.g. conjugated with a nucleotide, and then transferred to a glycosyl acceptor. In humans, CMAS and sialyltransferases (STs), retain the enzyme promiscuity necessary to efficiently conjugate exogenous Neu5Gc into CMP-Neu5Gc and subsequently incorporate the xenogenic Sia into a “xeno-autoantigen” glycan structure. However, the pseudogenization of the human CMAH gene occurred relatively recently, since the divergence of humans and chimpanzees. Compared to Neu5Gc, the efficiency of Sia activating and transferring enzymes towards Kdn seemed to be much lower, since only trace amounts of glycan-conjugated Kdn were observed in human and mouse cells cultured in high mannose media compared to efficient incorporation of Neu5Gc present in trace amounts in fetal calf serum supplemented media. In contrast, Kdn is

13 abundantly found in fish glycoconjugates based on a Cmas paralogue that has evolved preferential activity towards Kdn. Using a previously published expression construct (78), we show that in vitro expression of the Kdn-active zebrafish Cmas enzyme forced expression of small amounts of Kdn-containing cell surface glycans also in HEK293A cells (Figure 7, Supplemental Table 2-5), which normally excreted excess Kdn to maintain a constant cytosolic Kdn concentration (Figure 5, Supplemental Table 2-2). Free Neu5Ac or Kdn are synthesized by NANS from ManNAc-6P or Man-6P and phosphoenolpyruvate, respectively. In vitro analyses showed that Kdn biosynthetic activity is based on methionine or leucine at position 42 in human NANS. In line with glycosidically- conjugated Kdn, amphibia and fish NANS harbors methionine at position 42 (M42). Mouse NANS harbors the less active threonine (T42) and shows low Kdn biosynthesis in vitro (74). However, synthesis of free Kdn was observed in mouse cell culture in a high mannose media, indicating residual enzymatic activity on Man-6P. Phylogenetic analyses showed that the common vertebrate ancestor harbors the Kdn metabolizing M42 which is maintained mainly in non-mammalian classes and probably got lost during placentation in mammals (Figure 6). Reversion to methionine in Placentals occurred independently by convergent evolution in Primates and Artiodactyla. Given these phylogenetic occurrences of Kdn biosynthetic enzymes, it is tempting to speculate that these orders might be more prone to a mannose-rich diet and/or lack microbiota capable of mannose uptake. Free Sias are catabolized by action of N-acetylneuraminate pyruvate lyase (NPL) in vivo. However, human NPL has 5-fold lower activity on Neu5Gc in comparison to Neu5Ac and no detectable activity on Kdn (88), thus favoring the persistence of free Kdn. Interestingly, the distribution of Kdn in tissues of fish such as Atlantic salmon vary between skin and intestinal mucins; O-glycan structures showed that Neu5Ac, Neu5Gc, and Kdn are distributed in skin mucin, however, intestinal mucin contains only Neu5Ac (89, 90). Perhaps different host-pathogen interactions between aquatic and terrestrial habitats and/or constant exposure to hydrophilic aqueous environments produced different evolutionary pressures on Sia presentation. In this study, we explored why conversion of mannose into Kdn has been conserved in mammals like humans, despite lack of conclusive evidence of Kdn presence in sialoglycoconjugates. Based on our human experiments, we consider that mannose is handled like glucose in the kidney, being reabsorbed in the tubules, as previously reported (83, 91, 92),

14 implying that mannose is biologically “valuable”. However, excess mannose could also be excreted into urine, just as occurs with excess glucose in diabetic patients (“directly” mannose control) (Figure 3A). Moreover, phylogenetic analysis of vertebrate Kdn synthesis together with in vivo oral mannose administration and in vitro mannose feeding studies suggested that excess exogenous mannose is transported into the cytosol, then metabolized to Kdn through the conserved metabolic pathway, excreted from the cells, and finally eliminated from the body through the urine (“indirectly” mannose control). We confirmed by mannose supplementation in the media of different human (HEK293A, HUVEC, BJAB, HK2), as well as mouse (PMI) cell lines that mammalian cells very efficiently excrete cytosolic Kdn into media (Supplemental Table 2-2). Furthermore, Man-6P was not detectable in human serum and urine even after mannose ingestion (Supplemental Figure 3), consistent with our hypothesis that Kdn synthesis helps minimize buildup of toxic Man-6P in cytosol under conditions of mannose excess. However, we speculate that this is not the only reason. For example, mannose is 5 times more reactive in detrimental non- enzymatic protein glycation than glucose (93), it therefore needs to be regulated. Also, we have recently found that free Kdn can compete with host Neu5Ac for uptake by commensal and/or pathogenic bacteria, affecting their interaction with host immunity (94). Overall, our work can help explain the evolutionary conservation of Kdn biosynthesis throughout higher vertebrate phylogeny. Even though higher vertebrates, including humans, do not utilize Kdn in glycoconjugate production, Kdn synthesis remains a critical metabolic pathway that buffers the potentially toxic concentration of cytosolic Man-6P/Fru-6P. Moreover, humans produce anti-Kdn antibodies, probably due to the presentation of Kdn-containing antigens originating from symbiotic flora, as has been observed with human anti-Neu5Gc antibodies (14, 94). Our findings on the Kdn-mannose pathway (Figure 1) may contribute to future understanding of the exact pathways by which Kdn is taken up into cells and tissues, and by which humans produce anti-Kdn antibodies. Ultimately, deciphering the role of Kdn metabolism may serve to understand of human diseases, including congenital disorders of glycosylation involving PMI, NANS, and NPL (88, 95, 96), as well as ESRD. It also remains possible that small amounts of conjugated Kdn may appear in tissues under pathological conditions like ESRD with high free Kdn levels and mediate inflammatory reactions with circulating anti-Kdn-glycan antibodies. If human cells are forced to incorporate small amounts of Kdn into surface glycans (e.g., under

15 conditions of excess Kdn accumulation such as acute or chronic uncontrolled renal failure), low levels of cell surface accumulation could result in reactivity with such antibodies.

16 Methods

Human subjects - ESRD patients and control serum samples. Sera from ESRD patients were collected just prior to weekly dialysis. Control samples were collected from healthy volunteers. All samples were anonymized, numbered and kept at -80 ℃ before analysis.

Human mannose ingestion test. Five informed and consented healthy volunteers ingested 0.2 g mannose/kg body weight dissolved in 500mL of drinking water, as previously tested (47). Blood samples (up to total 60 mL/person) were collected by venipuncture at 0, 1, 2, 4, 6 and 8 hours. Urine samples were collected as near as possible to the aforementioned times. All samples were analyzed for free and conjugated Kdn, Neu5Gc and Neu5Ac, as well as for mannose.

Cell culture and transfection. HEK293A cells (ThermoFisher) were cultivated in RPMI1640 media with 10% FBS; HUVEC cells (ATCC , USA) in EBM2 media (Lonza, Inc); BJAB K20 and BJAB K88 cells (kindly provided by Stephan Hinderlich and the late Werner Reutter, Beuth University of Applied Sciences, Berlin) in RPMI1640 media with 10% FBS; PMI WT and PMI KO cells in DMEM containing 1g/L glucose, 10% FBS; HK2 cells in Keratinocyte Serum Free Media supplemented with Bovine Pituitary Extract and recombinant Epidermal Growth Factor. Transient transfection of Cmas1 and Cmas2 (and empty vector) was performed using PEI at a final concentration of 18 µg/100 cm tissue culture dish and 8 µg plasmid DNA following standard protocols. The transfection media was removed after 4.5 hours and the cells incubated in complete media with or without added mannose at indicated concentrations.

Cell viability measurement. Cell viability was measured using Real Time-Glo MT Viability Assay (Promega, Inc) according to instruction manual. Cells were incubated in culture media with various concentrations (25 µM - 200 mM) mannose or N-acetylglucosamine, which was used as control for osmolarity effects. MT Cell viability substrate and NanoLuc enzymes were also added to the culture and the viability monitored by reading luminescence at 0, 12, 24, 36, 48, 60, 72, and 84 hours.

Cell fractionation and sodium borohydride treatment. Cell fractionation and borohydrate treatment was done as previously reported (64) and detailed in the supplemental section.

17 Detection of serum mannose level by GC-MS. 100 µL of serum sample was spiked with 1µg of 13 C6-mannose as internal standard and 400 µL of cold acetonitrile (-20˚C) was added to precipitate the proteins. The sample was vortexed and kept on ice for 30 min to ensure complete precipitation. Sample was then centrifuged at 14,000 rpm at 4˚C for 7 min and the supernatant was removed and dried using speed vac and lyophilizer. The dried samples were added Tri-Sil reagent and incubated at 80˚C for 30 min. The reaction tube was then cooled to room temperature (RT) and excess reagent was removed by drying the samples. Tri-sil derivatized sample was then dissolved in hexane and

12 13 injected on GC-MS. Quantification was done by comparing the intensities of sample C6 and C6 mannose in the samples.

Detection of mannose in urine samples by GC-MS. Urine samples were centrifuged at 14,000 g for 5 min at 10°C to remove any insoluble material. 1 mL of urine sample was spiked with 1µg of 13 C6-mannose as internal standard, passed over pre-conditioned LC-NH2 SPE tubes (Supelco) and flow through was collected. The LC-NH2 SPE tubes were further washed with 1 mL of ultra-pure water and the dried flow through were then re-dissolved in 1 mL water transferred in screw capped glass reaction tube and dried in lyophilizer. 250 µL of Tri-Sil HTP reagent (Thermo Scientific) was added on the dried sample, sonicated (10-15 sec) and reacted at 80°C for 30 min. Tri-sil derivatized samples were then extracted by adding 1.5 mL of hexane followed by sonication, vortex and filtration using glass wool packed glass Pasteur pipette. The filtrate was dried and taken in GC-MS autosampler vial. The GC-MS analysis was done in EI mode using Restek-5MS glass capillary column (30 mL; 25 mm, i.d.). Identification of mannose was done from the retention time of Man on the column and quantified by comparing the ratio of ion intensities (m/z of 204 vs 12 13 206) of corresponding fragment mass from C6 and C6 mannose.

Detection of serum/urine sialic acid level by HPLC. 250µL serum were spun at 20,000g for 10 min, filtered and the DMB-derivatized samples were analyzed in HPLC as before (97). 400 µL of cold acetonitrile (-20˚C) was added to 100 µL urine sample and kept on ice for 30 min. Sample was then centrifuged at 14,000 rpm at 4˚C for 7 min and the supernatant was passed through a 3K spin filter. The flow-through was dried, DMB-tagged and detected using HPLC-FL under isocratic solvent condition (same as Sia-tagging).

18 Flow cytometry. Cells were blocked with 1% BSA in PBS and stained with mouse anti-Kdn antibodies on ice for 30 min. Following primary antibody, cells were washed and stained with APC-conjugated anti-mouse antibody (BioLegend; Poly4053). Anti-FLAG-F3165 (Sigma Aldrich) was used for population measurement for HEK293A cells with Cmas transfection. Acquisition and analysis were using BD FACSCalibur and FlowJo respectively.

Sequence alignments, tests for selection and phylogenetic tree. Coding nucleotide and amino acid sequences of mannose metabolism genes and proteins were obtained from NCBI nucleotide database and NCBI protein database. Nucleotide alignments and percent identity were produced using NCBI BLAST tool. Protein sequences were aligned using Clustal Omega program accessed via UniProt.org. Nucleotide alignments were used to analyze the ratio of nonsynonymous (Ka) to synonymous (Ks) substitution, evaluated with an online calculator

(http://services.cbu.uib.no/tools/kaks). The Ka/Ks ratio is representative of purifying selection on a coding sequence, as a Ka/Ks <1 indicates that there is pressure to conserve the amino sequence relative to the background mutation rate. Maximum likelihood tree was constructed using General Time Reversible model (98). The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying the Maximum Parsimony method. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.6749)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 21.44% sites).

Detection of anti-Sia antibodies in pooled human Intravenous immunoglobulin (IVIg). IVIg was used for the detection of anti-Sia antibodies (Kdn, Neu5Gc, Neu5Ac). Briefly, 96-well plate (CoStar) was coated overnight with 100 µg/mL of streptavidin at 4 ℃ and washed with PBS containing 0.1 % Tween 20 (PBST). The chemoenzymatically synthesized, defined glycans were used to determine the presence of antibodies against specific glycan structure. The glycan epitopes used were Neu5Acα2-3Galβ1-4GlcNAc-, Neu5Gcα2-3Galβ1-4GlcNAc-, Kdnα2-3Galβ1- 4GlcNAc- and unsialylated Galβ1-4GlcNAc- (negative control). Each of these glycans were biotinylated. Following streptavidin coating, the wells were further blocked with PBST for 1.5 hour at RT and coated with 500 ng of respective biotinylated glycan per well for 2 hours. Following incubation, the wells were washed with PBST and incubated with IVIG (1:250 dilution) for 1.5 hours at RT. Finally, the wells were washed and incubated with HRP-conjugated goat anti-human

19 IgG (BioRad, Cat.172-1050) antibodies (1:10000 dilution) for 1 hour, washed and visualized at 490 nm following HRP substrate addition. For quantification of intensity resulting from the specific antibodies against Neu5Ac/Neu5Gc/Kdn epitope and not from any nonspecific antibodies against the underlying glycan structure, the absorbance of the unsialylated conditions (wells coated with Galβ1-4GlcNAc- glycans) was subtracted from the corresponding values of the sialoglycan- coated wells.

Statistical analysis. Data were analyzed by t-test and ANOVA as indicated in figure legend and presented as mean (SD) using Prism software (version 9; GraphPad Software). P values less than 0.05 were considered significant.

Study approval. Human sample collection was approved by Human Research Protections Program, UC San Diego (UCSD HRPP) (111279XX). Informed consent was obtained prior to sample collection Ethical approval for human mannose ingestion study was obtained from UCSD HRPP (172014X).

Author contributions

Conceptualization, A.V.; Methodology, Ku.Ka., S.S., S.D., A.S., B.C., A.M.K., and A.V.; Validation, S.S., M.V.; Formal Analysis, Ku.Ka., S.D., M.V., A.S., Sh.Si., B.C., and A.M.K.; Investigation, Ku.Ka., S.S., S.D., M.V., A.S., Sh.Si., B.C., and A.M.K.; Resources, I.S., C.S., Ke.Ki., K.S., H.H.F., A.M.K., and A.V., Writing – Original Draft, Ku.Ka., and A.V.; Writing – Review & Editing, Ku.Ka., S.S., S.D., M.V., X.C., H.H.F., A.M.K., and A.V.; Visualization, Ku.Ka., S.S., M.V., Sh.Si., A.S. and A.M.K.; Supervision, H.H.F., A.M.K., and A.V.; Project Administration, A.V.; Funding Acquisition, H.H.F., and A.V.; Ku.Ka. - Methodology, Formal analysis, investigation, visualization, original draft writing, review and editing; S.S. - Methodology, Validation, investigation, visualization, draft review and editing.

Acknowledgments

This work was supported primarily by NIH Grant R01GM32373 (to A.V.) and by the Rocket Fund and R01DK99551 (to H.H.F.). M.V. was supported in part by a Ruth L. Kirschstein Institutional

20 National Research Award from National Institute for General Medical Sciences, T32 GM008666 and Training Grant DK007202.

21 References

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29 HO OH HO O HO 2 O PO3 Man-1P Not typically produced in PMM2 human cell OH 2 2 OH HO OH ATP ADP O P O OH PEP O3P O OH OH OH HO HO OH 3 OH OH O CMP HO O HO O O CO O OH HO HO 2 HO CO2 O HO HK NANS NANP CMAS HO CO2 HO OH HO HO Man Man-6P Kdn-9P Kdn CMP-Kdn PMI OH HO 2 OH O CMP O3P O O OH HO H O N CO2 Glyco- HO OH HO conjugates OH O Fru-6P CMP-Neu5Gc

CMAH

OH HO HO OH O CMP HO O HO O AcHN CO2 AcHN NPL O UDP HO UDP-GlcNAc CMP-Neu5Ac GNE CMAS 2 2 OH OH NHAc O P O HO NHAc O3P O PEP 3 OH OH OH OH HO O O HO HO OH HO O CO2 O CO HO GNE NANS AcHN NANP AcHN 2 OH HO HO ManNAc ManNAc-6P Neu5Ac-9P Neu5Ac

Figure 1. Metabolic pathway associated with mannose and sialic acids. Mannose is associated with sialic acid metabolism. Mannose is phosphorylated to mannose-6- phosphate (Man-6P) by hexokinase and adenosine triphosphate (ATP) as cofactor. Man-6P is converted to 2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (Kdn) with N- acetylneuraminate synthase (NANS) and N-acetylneuraminic acid phosphatase (NANP), and Kdn can be converted to cytidine monophosphate (CMP)-Kdn with CMP N-acetylneuraminic acid synthetase (CMAS) in some species. Man-6P can also be transformed into mannose-1-phosphate (Man-1P) by phosphomannomutase 2 (PMM2). Man-6P is also converted to Fructose-6-phosphate (Fru-6P) by phosphomannose isomerase (PMI). Fru-6P is transformed into uridine diphosphate N- acetylglucosamine (UDP-GlcNAc), then converted N-acetylmannosamine (ManNAc)-6- phosphate with UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). ManNAc-6P is converted into N-acetylneuraminic acid (Neu5Ac) via NANS and NANP. N-acetylneuraminate lyase (NPL) regulates Neu5Ac concentration by mediating the reversible conversion into ManNAc and pyruvate, but human NPL does not work on Kdn. Neu5Ac is converted into CMP-Neu5Ac with CMAS and CMP-Neu5Ac is converted into CMP-N-glycolylneuraminic acid (Neu5Gc) by CMP-

30 Neu5Ac hydroxylase (CMAH). Humans cannot synthesize Neu5Gc due to inactivation of CMAH gene about 2-3 million years ago.

31 A 4×107 Control ** ESRD

3×107

2×107 ** 7 ** 1×10 ** 6×106

6 ** Area counts (millions) counts Area 4×10 ** ** ** ** ** 2×106 ** ** ** ** ** ** ** 0

Kdn Kdo Neu5GcNeu5Ac unknown3unknown4unknown5unknown6 unknown7unknown8unknown9 unknownunknown 1 2 Neu5,7Ac2 Neu5,8 Ac2Neu5,unknown10 9Ac2 unknown11unknown12unknown13unknown14 Neu5Gc9Ac Neu5,8,9Ac3 Neu5, 7, 9 Ac3 B C D 5 ** 25 ** 150 ** 4 20 M) M) µ

µ 100 M)

µ 3 15

2 10 Kdn ( 50 Neu5Ac ( 1 5 Mannose (

0 0 0 Control ESRD Control ESRD Control ESRD

Figure 2. DMB-HPLC profiling of hemodialysis patients’ serum samples in comparison to healthy serum samples. Serum samples from (A) Healthy volunteers (Control) (n=14), End-stage of renal disease (ESRD) patients on hemodialysis (n=58) were analyzed by DMB-HPLC. Each dot represents each serum. Serum free (and CMP-) form of (B) Kdn and (C) Neu5Ac were calculated with DMB-HPLC. (D) Serum mannose level (normal range, 40-80 µM) measured by GC-MS. Control (n = 8) vs. ESRD (n = 16). Shown are mean (SD), statistics using 1-way ANOVA (A) and unpaired t-test (B-D). **p < 0.01.

32 A B 300 serum 40 4

urine Urine mannose (

M) ** * µ ** * 30 3 200 * Mole) * µ 20 2

100 µ 10 M) 1 Urine Kdn ( Serum mannose (

0 0 0 0 1 2 4 6 8 0 1 2 4 6 8 Time (hrs) Time (hrs)

Figure 3. Human mannose ingestion test reveals increase of urinary Kdn. Mannose (0.20 g/kg body weight) were used for human ingestion test (healthy volunteers, n=5). (A) Time course (0-8 hours after mannose ingestion) for serum and urine mannose levels were measured by GC-MS. (B) Urinary Kdn levels were evaluated by HPLC. Shown are mean (SD), 2-way ANOVA, *p<0.05, **p < 0.01.

33 Mannose GlcNAc A 6 B 7 7 1.2×10 3.2×10 1.2×10 0 mM 6 6 5 mM 9.0×10 6 9.0×10 2.4×10 25 mM HEK293A 6.0×106 6.0×106 50 mM 6 6 6 3.0×10 1.6×10 3.0×10 100 mM 200 mM

Luminescence (RLU) 0.0 0.0 0 12 24 36 48 605 72 84 0 12 24 36 48 60 72 84 Time8.0 ×(hrs)10 C 6 D 7 1.0×107 3.2×10 1.0×10 0 mM 8.0×106 0.0 8.0×106 5 mM 60 12 24 36 48 60 72 84 6.0×106 2.4×10 6.0×106 25 mM HUVEC 4.0×106 4.0×106 50 mM 6 2.0×106 1.6×10 2.0×106 100 mM 200 mM

Luminescence (RLU) 0.0 0.0 0 12 24 36 48 605 72 84 0 12 24 36 48 60 72 84 Time8.0 ×(hrs)10

E 6 F 3.2×106 3.2×10 3.2×106 0.0 0 mM 6 6 5 mM 2.4×10 60 12 2.424×1036 48 60 72 84 2.4×10 25 mM BJAB K88 1.6×106 1.6×106 50 mM 5 5 8.0×10 1.6×106 8.0×10 100 mM 200 mM

Luminescence (RLU) 0.0 0.0 0 12 24 36 48 605 72 84 0 12 24 36 48 60 72 84 Time8.0 ×(hrs)10 G 6 H 6.0×106 3.2×10 6.0×106 0.0 0 mM 6 6 5 mM 4.5×10 60 12 4.524×1036 48 60 72 84 2.4×10 25 mM BJAB K20 3.0×106 3.0×106 50 mM 6 6 1.5×10 1.6×106 1.5×10 100 mM 0.0 200 mM Luminescence (RLU) 0.0 0 12 24 36 48 605 72 84 0 12 24 36 48 60 72 84 Time8.0 ×(hrs)10 I 6 J 4.0×106 3.2×10 4.0×106 0 mM 0.0 5 mM 3.0×106 6 3.0×106 2.4×10 0 12 24 36 48 60 72 84 25 mM PMI WT 2.0×106 2.0×106 50 mM 1.0×106 1.6×106 1.0×106 100 mM 200 mM

Luminescence (RLU) 0.0 0.0 0 12 24 36 48 605 72 84 0 12 24 36 48 60 72 84 Time8.0 ×(hrs)10 K L 2.5×106 6 2.5×106 2.5×10 25 µM 2.0×106 0.0 2.0×106 60 12 24 36 48 60 72 84 1 mM 1.5×106 2.0×10 1.5×106 PMI KO 10 mM 1.0×106 1.0×106 6 15 mM 5.0×105 1.5×10 5.0×105 30 mM 0.0 0.0 Luminescence (RLU) 6 0 12 24 1.036 48×1060 72 84 0 12 24 36 48 60 72 84 Time (hrs) 5.0×105

0.0 Luminescence (RLU) Figure 4. Toxicity of excess mannose in human0 12 and24 mouse36 48 cells60 in a72 dose84 dependent manner. Time (hrs) Continuous cell viability assay (0-84 hours) evaluated mannose toxicity on various human and

34 mouse cell lines including mutants of Kdn associated metabolic enzymes. Mannose feeding concentration was set in 0-200 mM and same concentration of GlcNAc was used as control for hyperosmolarity effect. (A), (B) HEK293A; (C), (D) HUVEC; (E), (F) BJAB K88 (GNE WT); (G), (H) BJAB K20 (GNE KO); (I), (J) PMI WT cells; (K), (L) PMI KO cells. PMI KO cells were always maintained in mannose (25 µM) supplemented media for their survival during GlcNAC feeding (L). Representative data from two independent experiments. UDP-GlcNAc 2- epimerase/ManNAc kinase, GNE; Phosphomannose isomerase, PMI; RLU, Relative luminescence unit.

35 A HEK293A B BJAB K88 C BJAB K20 D PMI WT E PMI KO 1.0 20 100 * 1.0 1.0 0.8 0.8 0.8 15 80 0.6 60 0.6 0.6 10 0.4 40 0.4 0.4 5 0.2 20 0.2 0.2

Kdn (relative to Neu5Ac) 0.0 0 0 0.0 0.0 0 15 0 15 0 15 0 15 0.025 0.25 1 Mannose (mM) Mannose (mM) Mannose (mM) Mannose (mM) Mannose (mM)

F HEK293A G BJAB K88 H BJAB K20 I PMI WT J PMI KO 3 3 3 3 3

2 2 2 2 2

CMP-Kdn 1 1 1 1 1 (relative to Neu5Ac) 0 0 0 0 0 0 15 0 15 0 15 0 15 0.025 0.25 1 Mannose (mM) Mannose (mM) Mannose (mM) Mannose (mM) Mannose (mM)

Figure 5. Kdn/Neu5Ac ratio increases in cytosolic fractions after mannose feeding in human and mouse cell types. Cytosolic free (and CMP-) Kdn (A-E) and CMP-Kdn (F-J) were measured by DMB-HPLC: (A), (F) HEK293A; (B), (G) BJAB K88 (GNE WT); (C), (H) BJAB K20 (GNE KO); (D), (I) mouse embryonic fibroblast PMI WT cells; (E), (J) PMI KO cells. CMP form of sialic acids were measured by DMB-HPLC after NaBH4 treatment. Mannose concentration was set at 0 or 15 mM except PMI KO (0.025, 0.25, and 1 mM). UDP-GlcNAc 2-epimerase / ManNAc kinase, GNE; Phosphomannose isomerase, PMI. Shown are mean (SD), unpaired t-test, *p<0.05.

36

Figure 6. A single amino acid in sialic acid 9-phosphate synthase that alters the rate of Man

37 to Kdn conversion is restored independently by convergent evolution in two mammalian lineages including humans. Phylogenetic tree topology of vertebrate NANS nucleotide sequences obtained by using the Maximum Likelihood method. The tree was rooted on non-mammalian vertebrates (●) and is not drawn to scale. Human NANS with Met or Leu at position 42 show Kdn- 9P synthase activity. Species with Met or Leucine at the position aligned to M42 in human NANS are shaded in light blue, and species with threonine (T42) or valine (V42) are shaded in light and dark grey, respectively. Predicted nucleotides encoding the aa at the respective position in the ancestors are given at the nodes. Human NANS is shown in red. Latin species names are italicized; common names are given in uppercase letters. Mammals are further classified in orders, all vertebrates in classes.

38 Sample % Mean:FL4-H 100 100 Sample % Mean:FL4-H 7 68.7 6 15 70.3 5.06 5 60.2 4.16 13 73.5 3.82 3 62.8 5.95 80 80 11 69 5.07 1 63.9 6.44 9 65.9 5.97

60 60 % of Max % of Max 40 40

20 20

0 0 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 FL4-H FL4-H BJAB K20 BJAB K88

Sample % Mean:FL4-H 100 Sample % Mean:FL4-H 23 91.3 19.4 100 31 89.3 25.6 21 92.1 6.72 29 88 12.4 19 92.4 17.5 100 80 Sample % Mean:FL4-H Sample % 27Mean:FL4-H88.4 35.5 17 93.7 9.14 100 80 7 68.7 6 15 70.3 255.06 92 11.8 5 60.2 4.16 13 73.5 3.82 60 3 62.8 5.95 80 80 60 11 69 5.07 1 63.9 6.44 9 65.9 5.97 % of Max

40 % of Max 60 60 40 % of Max 20 % of Max 40 40 20

20 0 0 1 2 3 4 20 0 10 10 10 10 10 0 1 2 3 4 FL4-H 10 10 10 10 10 PMI WT FL4-H 0 0 PMI KO 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 FL4-H FL4-H BJAB K20 BJAB K88

SampleSample% % Mean:FL4-HMean:FL4-H 100100 Sample % Mean:FL4-H 238 91.371.319.412.4 100100 Sample % Mean:FL4-H 1631 73.389.3 8.7425.6 216 92.171.26.727.95 100 Sample % Mean:FL4-H Sample % Mean:FL4-H 1429 7088 8.7512.4 8080 100 194 92.466.717.510.4 7 68.7 6 1227 66.188.4 1135.5 172 93.768.59.148.38 808015 70.3 5.06 5 60.2 4.16 13 73.5 3.82 1025 65.792 9.2811.8 80 3 62.8 5.95 6060 80 11 69 5.07 1 63.9 6.44 60609 65.9 5.97 % of Max 60 % of Max % of Max 4040 60 % of Max 4040 % of Max % of Max 40 2020 40 2020

20 0 0 20 Bound Neu5Gc 0 0 Bound1 1 Neu5Gc2 2 3 3 4 4 00 1010 1010 1010 1010 1010 00 11 22 33 44 in HEK293 A membrane in HEK293 AFL4-H membraneFL4-H 1010 1010 1010 1010 1010 FL4-H 26 0 Bound1 mM Neu5Gc Man isotype 26 PMIBJABK20 WT 0 1 mM Man isotype FL4-H Bound Neu5Gc 0 1 2 3 4 Bound Neu5Gc Bound Neu5Gc BJABPMI KO K88 Bound Neu5Gc A 10 25 10µM Man 10Kdn3g 10 10 B Bound Neu5Gc 100C 101 25 µ10M2 Man Kdn3g103 104 D in HEK293 A membraneFL4-H in HEK293 A membrane in inHEK293 HEK293 A Amembrane membrane Bound Neu5Gc inin HEK293HEK293 AA membranemembrane FL4-H 24 26 BJAB K20 2510 mM µ mMM Man Man isotype isotype 24 151 mM mM Man ManKdn3g isotype 26BJAB K88 Bound2525 µ MNeu5GcµM Man Man isotype isotype 1 mM Man Kdn3g 26 26 20180130 Workspace.join HEK2931 mM A membraneMan isotype 2626 Layout: histogram 11 mMmM ManMan isotypeisotype 1 mM Man isotype 25 µM Man Kdn3g in HEK293 A membrane 25 µM Man Kdn3g 26 25 µM ManMan Kdn3g Kdn3g 1 mM Man isotype 26 2525 µµM Man Kdn3g 1 mM Man isotype 22 0 mM Man Kdn ab 22 15 mM Man Kdn ab 24 25 µM Man Kdn ab 125 mMµM Man Man isotype Kdn ab 24 24 25 µM Man isotype 24 25 µM Man isotype 20180130 Workspace.jo 25 µM Man isotype 20180130 Workspace.jo25 µM Man Kdn3gLayout: histogram Layout: histogram 24 Cmas25 µM 2 Man isotype 2018013024 Workspace.jo25 µM Man Kdn3g Cmas25 µM 2ManLayout: isotype histogram 24 25 µM Man isotype 20 24 20 25 µM Man isotype 22 22 22 BJAB K20– kdn3G 22 BJAB K20 – kdn8kdn PMI KO – kdn3G PMI KO – kdn8kdn 22 100 22 Sample % Mean:FL4-H Cmas 2 Cmas 2 100 Sample % Mean:FL4-H 22 SampleSampleCmas% %Mean:FL4-H 2 Mean:FL4-H Sample % Mean:FL4-H Sample Gate % Mean:FL4-H 100 100 Cmas23 2 91.3 19.4 100 100 Sample100 % Mean:FL4-H Membrane Sias 22 Cmas 2 Membrane Sias 7 68.7 6 20 8 24Cmas71.388.912.4 2 13.3 100 Cmas31 289.3 25.6 Sample % Mean:FL4-H 2018 2018 21 92.1 6.72 15 70.3 5.06 32 PMI KO 647 84.3 21 (pmoles / mg protein) 20 (pmoles / mg protein) 22 90.5 8.43 16 73.3 8.74 20 Cmas5 2 60.2 4.16 20 6 71.2 7.95 13 73.529 3.8288 12.4 30 PMI KO 647 84.1 12.6 80 20 19 92.4 17.5 14 70 8.75 80 3 62.8 5.95 Membrane Sias 20 91.6 10.9 27 88.4 35.5 Membrane Sias Membrane Sias 4 66.7 10.4 28 PMI KO 89.4 49 18 80 18 80 17 93.7 9.14 18 80 80 1180 69 5.07 16 20 16 Membrane Sias (pmoles / mg protein) 18 90.5 8.78 80 12 66.1 11 Membrane Sias (pmoles / mg protein) (pmoles / mg protein) 1 63.9 6.44 18 2 68.5 8.38 9 65.925 5.9792 11.8 26 PMI KO 647 90.2 12.5 Membrane Sias (pmoles / mg protein) 18 18 Membrane Sias 10 65.7 9.28

(pmoles / mg protein) 18

(pmoles / mg protein) 60 HO (pmoles / mg protein) 16 16 0HO mM Membrane Sias HO HO 16 60 0 mM OH 15 mM 18 60 OH 60OH 15 mM 16 60 60 60

(pmoles / mg protein) OH 60 16 16 Mannose % of Max 16 OH Mannose 0 mM 0 mM OH OH % of Max 0 mM 0 mM 15 mM % of Max % of Max % of Max 15 mM OH % of Max 15 mM 16 40 % of Max 15 mM HO 40 % of Max HO CO HO 40 CO HO CO 40 CO 40 40 40 Mannose 0 mM 2 Mannose Mannose 40 0 mM 2 2 15 OmM 2 O 0 mM O 15 mM O HOHO HO 15 mM HO HOHOHO 0 mM HOHO HO OH HO OH 20 OH HO15 mM OH 20 20 Mannose 20 20 20 20 Mannose Mannose 20 OH OH OH Kdn HO HOMannoseKdn OH HO KdnKdn HO H Kdn KdnHKdn Normalizedcounts KdnH H CO N 0 CO CO N 0 CO N 0 N 0 0 2 0 0 O 100 2 10HO1 102 103 2 104 0O 21 HO 2 O3 4 HO 0 0 1 HO 2 O 3 HO4 010 110 210 HO 3 10 4 100 0 HO1 1 2 2 3 3 4 4 0 1 2 3 4 O HO 10 10 10FL4-H 10 10O HO10 10 O10 HO10 1010 10 10 10 10 10 10 1010 10 0 10 1 10 2 10 3 10 4 10 15 mM Kdn3g O HO GlcNAc 15 mMOH Kdn3gFL4-H HO OH 10 10 10 10 10 GlcNAc GlcNAcPMI WT OH GlcNAcFL4-H OH FL4-H 15 mMHOKdn3gFL4-H FL4-H FL4-H Fluorescence intensity PMI WT 647 OH HOOH FL4-H 1515 mM Kdn3gisotype BJAB K20 OH BJABK20OH 1515 mMmMKdn3gisotype OH BJAB K88PMI KO 15 mM Kdn3gOH OH PMI KO 647 15H mM isotype BJAB K88 Neu5Gc H Neu5GcH Neu5GcH 150 mM mMKdn3gisotype Neu5Gc GalNeu5Gc CO Gal Neu5Gc150 COmM mMKdn3gisotypeNNeu5Gc COOH 15N mM isotypeCOOH 7 Gal Gal E N Neu5Gc N 0 mM Kdn3gOH 2 4×10 2 2 KdnHOO 2 CO O 00 mM Kdn3gisotype O Neu5Ac O 00 mMmMKdn3gisotype O 0 HOmMHOKdn3gisotypeHO CO CO Kdn R 15R mM Kdn3gO HO R O HO O HO 2 OH 2 15 mM Kdn3g R OH 15 OHmM Kdn3g OHO 0 mM isotype O 2 0 mM isotype GlcNAc GlcNAc GlcNAc 0 mM isotypeGlcNAc HO HO O HO 15 mM isotypeKdn3g OH HO HOHO OH HO HO7 15 mM isotype 1/30/18 2:25 PM Page 1 of 8 15 mM isotype(FlowJo v9.7.6) OH HO3×10 100 Sample % Mean:FL4-H Gc 015 mM mMKdn3gisotype Neu5Ac 100 Neu5Ac Sample % Mean:FL4-H Ac 0 mM Kdn3g Gal Neu5AcNeu5Ac GalNeu5Ac 23 91.3 19.4Gal Neu5Ac HONeu5Ac OH OH OH Gal Neu5Ac0 mM Kdn3g KdnKdn KdnHKdnKdn HO HO H 31 89.3 25.6 0 mM Kdn3g 21 92.1 6.72 1/30/18 2:25 PM Kdn COPage 4 of 8 H1/30/18 2:257 PM CO (FlowJo v9.7.6) Page 8 of 8 (FlowJo v9.7.6) 0 mM isotype 0 mM isotype 1/30/18 2:25 PM Page 5 of 8 N (FlowJo v9.7.6) N 2N×10 29CO 88 12.4 10080 R Sample19 0 R%mM92.4Mean:FL4-Hisotype17.5 Sample % Mean:FL4-HO 2 HO 2 R 0 mMR isotype 100 100HO Sample27O% 2 Mean:FL4-H88.4HO35.5HO Sample Gate % Mean:FL4-H GlcNAc 8 17 71.393.712.49.14 GlcNAc 100 80 GlcNAcO HO O HOO HO HO GlcNAc GlcNAc 24 88.9 13.3 OH O HO 16 73.3 8.74OH 32 PMI KO 647 84.3 21 6 71.2 7.95 GlcNAcGlcNAc 25 OH 92 11.8 22 90.5 8.43 OH14 7 70 8.75 OH 30 PMI KO 647 84.1 12.6 4 66.7 10.4 mannose 1×10 OH 8060 20 91.6 10.9 HArea counts HEK293AGal empty vector80 Cmas1Gal 80Cmas260 Gal Neu5Gc Neu5Gc80 12 66.1 11 H H 28 PMI KO 89.4 49 Gal 2 68.5 8.38 Gal 18 Gal90.5Neu5Gc8.78 Neu5GcN Neu5GcCO N CO Gal Neu5Gc10 65.72 9.28 N CO 26 PMI KO 647 90.2 12.5 feeding O O 2 2 % of Max R R R O HO O R R % of Max 0 O HO 6040 60 R R OH O HO OH 60 40 GlcNAc GlcNAcGlcNAc60 OH % of Max

7 % of Max % of Max 1×104020 500000040 % of Max Neu5AcNeu5Ac Neu5Ac 40 20 Gal GalGal40 Neu5AcNeu5AcNeu5Ac F 0 mM mannose 4000000 G 15 mM mannose RH 0 mM mannose R I 15 mM mannose 20 0 R 20 GlcNAc20 GlcNAc 0 1 2 3 4 20 0 GlcNAc 10 10 10Ac 10 300000010 Ac 0 1 2 Ac3 4 Ac 4×107 FL4-H 7 4×10 7 10 10 10 10 4×107 6 4×10 FL4-H 5×10PMI0 WT Gal 0 PMI KO 0 GalGal 0 1 2 3 20000004 0 10 10 10 10 10 100 101 102 103 104 0 1 2 3 4 7 7 100 7101 102 103 104 10 710 10 10 10 3×10 FL4-H 3×10 FL4-H 3×10 R 3×10 R FL4-H R FL4-H BJABK20 PMI WT 647 1000000 BJAB K88 PMI KO 647 2×107 7 2×107 2×107 0 2×100 0 0 1×107 Gc 1×107 Gc 1×107 Gc 1×107 Gc Sample % Mean:FL4-H 100 Sample % Mean:FL4-H 0 8 71.3 12.4 100 0 0 0 16 73.3 8.74 6 71.2 7.95 Sample % Mean:FL4-H 14 70 8.75 10080 4 Kdn66.7 10.4 Kdn Sample GateKdn% Mean:FL4-H 100 12 66.1 11 242 88.968.513.38.38 80 32 PMI KO 647 84.3 21 10 65.7 9.28 22 90.5 8.43 30 PMI KO 647 84.1 12.6 20 91.6 10.9 8060 HEK293A (empty vector) 80 HEK293A28 (Cmas2PMI KO 89.4) 49 18 90.5 8.78 60 26 PMI KO 647 90.2 12.5 % of Max

6040 % of Max 60 40 % of Max % of Max 4020 40 Figure 7. Gene mutations or fish Cmas transfection20 can enforce production of the conjugated

200 20 0 1 2 3 4 0 form10 of Kdn.10 10 Mouse10 monoclonal10 anti-Kdn IgG 0 antibody,1 2 kdn3G3 recognizing4 (Kdn) ganglioside FL4-H 10 10 10 10 10 FL4-H BJABK200 0 0 1 2 3 4 BJAB K88 10 10 10 10 10 100 101 102 103 104 GM3; mouseFL4-H monoclonal anti-Kdn IgM antibody, kdn8kdnFL4-H recognizing α2,8-linked oligo Kdn PMI WT 647 PMI KO 647 were used for flow cytometry to analyze cell surface expression of Kdn. (A) and (B), BJAB K20

Sample % Mean:FL4-H (GNE100 KO); (C) and (D), PMI KO, with or without100 mannose feeding. (E)Sample EmptyGate % vector,Mean:FL4-H Cmas1 24 88.9 13.3 32 PMI KO 647 84.3 21 22 90.5 8.43 30 PMI KO 647 84.1 12.6 20 91.6 10.9 80 80 28 PMI KO 89.4 49 and Cmas2 were transiently18 expressed90.5 8.78 in HEK293A cells. Cells were harvested26 PMI KO 647 90.2 after12.5 36 hours mannose60 feeding (0 or 15 mM) then membrane 60fractions were prepared to analyze conjugated form % of Max % of Max 40 40 of Sia by DMB-HPLC analysis (Kdn, Neu5Gc and Neu5Ac). Representative HPLC peaks for 20 20 empty vector (F and G) or Cmas2 (H and I) transfected membrane samples: (F and H) 0 mM; (G 0 0 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 FL4-H FL4-H andPMI WT 647 I) 15 mM mannose fed. Representative dataPMI KO 647from two independent experiments.

39 A B OH HO HO OH HOHO HOHO HO HO HO HO OH HOHO HOHO CO OH CO2 OH CO2 HO CO OHOH COCO2 OHOH COCO2 OH 2 OHOH CO2 2 2 0.8 2 H H H H H H O OH O OH O OH OO OHOH O O OHOH O O OHOH HO N N HOHO N N N N HO HO HOHO HO HO HOHO HOHO HOHO O O OO O O0.6 KdnKdn Neu5Gc Neu5Ac KdnKdnKdnKdn Neu5GcNeu5Gc Neu5AcNeu5Ac Neu5Gc Neu5Ac Neu5GcNeu5Gc Neu5AcNeu5Ac 0.4 GlcNAc GlcNAc GlcNAc GlcNAcGlcNAc GlcNAcGlcNAc GlcNAcGlcNAc

Gal Gal Gal GalGal GalGal 0.2 GalGal Absorbance (490 nm) R R R RR RR 0.0RR Subtracting asialo-IgG signal Neu5Ac Kdn Neu5Gc

Figure 8. ELISA of anti-sialoglycan antibodies in human sera. (A) The chemoenzymatically synthesized, defined biotinylated glycans; Neu5Acα2-3Galβ1-4GlcNAc-, Neu5Gcα2-3Galβ1- 4GlcNAc-, Kdnα2-3Galβ1-4GlcNAc -biotin respectively. (B) Anti-Sia antibodies (Kdn, Neu5Gc and Neu5Ac) were detected in pooled normal human sera (Intravenous immunoglobulin; IVIg) samples (n=6 each). The absorbance of the wells coated with Galβ1-4GlcNAc- (negative control) glycan was subtracted from the corresponding sialoglycan containing wells to determine the relative absorbance resulting from the specific anti-Sia antibodies. Shown are mean (SD), 1-way ANOVA.

40 Table 1. Search of Glycan Databases for Glycosidically-linked Kdn.

Database Total Occurrence Glycans Glycans Occurrence of Reference number of of with with Kdn glycan Neu5Ac Neu5Ac Kdn structures

GlyGen 29290 Mammals 8963 none (99) (https://www.gly gen.org) GlyConnect 3435 Mammals, 371 35 Fish, (100) (https://glyconne Fish, Aves, Amphibians ct.expasy.org) Amphibian

UniCarb-DB 1118 Mammals 415 none (101) (https://unicarb- db.expasy.org) LipidBank 7009 Mammals, 100 17 Fish, (102) (http://lipidbank. Fish, Aves, Amphibians jp) Amphibia GlyTouCan 117832 Mammals, 6570 334 Fish, (103) (https://glytouca Fish, Aves, Amphibians n.org) Amphibia

41 Table 2. Conservation of Human Kdn biosynthesis pathway genes.

PMM2 PMI NPL NANS NANP GNE CMAS Ka/Ks

Homo 0.000 0.000 0.000 0.000 0.000 0.000 0.163 Mus 0.047 0.132 0.100 0.029 0.069 0.005 0.317 Gallus 0.115 0.329 0.265 0.038 0.166 0.023 0.347 Anolis 0.162 0.276 0.274 0.089 0.292 0.068 0.201 Xenopus 0.206 0.265 0.324 0.151 0.272 0.091 Danio 0.176 0.279 0.337 0.125 0.397 0.113 0.038# 0.264* % AA

Homo 100 100 100 100 100 100 100 Mus 92 85 87 95 94 99 94 Gallus 85 64 71 87 72 93 77 Anolis 79 67 69 85 68 92 84 Xenopus 69 62 54 78 59 86 63 Danio 73 63 57 79 50 81 60# 53*

% nt

Homo 100 100 100 100 100 100 100 Mus 88 87 86 89 86 88 88 Gallus 78 70 74 81 67 81 76 Anolis 75 71 71 77 72 80 78 Xenopus 67 67 64 74 74 77 68 Danio 71 69 66 74 71 68#

75*

Ka/Ks (top), percent amino acid identity (middle), and percent nucleotide identity (bottom), values of genes involved in the synthesis of Sia across vertebrate phylogeny: Homo sapiens (human); Mus musculus (mouse); Gallus gallus (chicken); Anolis carolinensis (green-anole

42 lizard), Xenopus laevis (african clawed-frog); Danio rerio (zebrafish). For zebrafish, Cmas1 gene is marked # and Cmas2 is marked*. The whole-genome duplication event that affected Cmas in Teleostei also produced a paralogue of Nans; however, expression and function of Nansb gene is unknown so for our phylogenetic analysis we focused on zebrafish Nansa (96).

43 Graphical abstract

Neu5Ac, Kdn Neu5Ac

-6P ↑ ATP ↑ Predominant Sialic Acids Man in Glycans Honeybee Syndrome Neu5Ac, Kdn Neu5Ac -6P ↑ Man Kdn Neu5Ac Neu5Ac, Neu5Gc Mannose Buffering in Mammals

44 Supplemental Materials and Methods

Detection of free α-ketoacids including sialic acids and by LC-MS. Analyses of α-ketoacids including Kdn, Neu5Ac, Neu5Gc (Supplemental Table 1) were done using LTQ-Orbitrap Discovery (Thermo Scientific) mass spectrometry attached to Ultimate3000 HPLC system (Thermo-Dionex) after tagging with DMB. The profiling of α-ketoacids was done using Phalanx C18 column (150mm x 1.0mm, 5µm) from Higgins Analytical. A step-gradient of solvents at a flow rate of 50 µL was used to get optimal separation between the sugar moieties. 5% aqueous methanol containing 0.1% formic acid was used as Solvent-A and mixture of 5% methanol, 45% water and 50% acetonitrile containing 0.1% formic acid was used as Solvent-B; the details of gradient is as follows. For 0-5 min mixture of 83% of solvent-A and 17% of solvent-B; for 5.1- 10min 80% of solvent-A and 20% of solvent-B; for 10.1 to 15min 75% of solvent-A and 25% of solvent-B was used; for 15.1-20 min 70% of solvent-A and 30% of solvent-B is used and finally from 20.1-25min 50% solvent-A and 50% solvent-B was used. The mass spectral data was obtained in positive mode.

Sequence alignments, tests for selection and phylogenetic tree. Evolutionary analyses of NANS were conducted in MEGA X (Kumar S, Stecher G, Li M, Knyaz C, and Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35:1547-1549). Information about sequences are given in Supplemental Table 4. Multiple alignment based on NANS nucleotide sequences was performed using MUSCLE with UPGMA algorithm.

Detection of Man-6P in human serum and urine by HPAEC-PAD. The frozen serum and urine samples were thawed and centrifuged at 3,000 rpm for 5 min at 4°C. Samples were divided into two 500 µL aliquots and one of them was added Man-6P (0.2 µg total per sample). Then the samples were filtered by Microcon-3 filter at 14,000 X g for 20 min at 4°C. Next, the flow through was passed over 1mL column packed with Dowex cation exchange resin (AG 50W-X8, 200-400 mesh). Then the resin was washed with 1 mL ultrapure water and the flow through was collected for Man-6P analysis using HPAEC-PAD (Dionex ICS-3000). Carbopac PA-1 column (Dionex CarboPac PA1 column 4 mm x 250 mm, 4μm, with a 4 mm x 50 mm Guard) was used for monosaccharide profiling with 100 mM NaOH and 250 mM NaOAc as gradient mixture.

45 Quantification was done by comparing with the known quantity of standard monosaccharides purchased from Sigma.

Cell fractionation and sodium borohydride treatment. Cells and media were collected after incubation with mannose. The media was centrifugated and 1.5 mL aliquot was passed filtered through a Microcon-10 filter and lyophilized. Cells were harvested with 10mM EDTA and washed with PBS; suspension cells were directly washed with PBS. Hereon all fractions are kept on ice.

The cell pellets were lysed using ice cold water which had been titrated to pH 7.5 with 1M NH4OH (for preservation of CMP-glycans) and sonicated with two 15 second bursts, with 15 second rest in-between. The homogenates were centrifuged at 100,000 g for 1 hour. The supernatant was collected, adjusted to 87.5% ethanol and centrifuged at 20,000 g for 10 min. The resulting supernatant was diluted to 10 % ethanol (using pH 7.5 water), frozen and lyophilized; this fraction was called FSF (Free-Sugar Fraction) containing Neu5Ac, Kdn, CMP-Neu5Ac, CMP-Kdn. The resulting pellet was called SPF (Soluble Protein Fraction) and was stored at -80˚C. The cell pellet was washed once with 3 mL cold water pH 7.5, sonicated, and centrifugation at 100,000 g for 1 hour. The membrane pellet was suspended in 200 µL water pH 7.5. Sodium borohydride treatment was performed on 25 µL of the FSF and media (after lyophilization, the dried material was dissolved in 200 µL ice-cold water). The sample was mixed with 25 µL of 0.5 M NaBH4, incubated at 37˚C for 15 min, and quenched with 25 µL 5 M acetic acid, for 5 min at 4˚C and 10 min at37˚C. Control samples which were not subjected to borohydride treatment, had 25 µL of water added, 25 µL 5 M acetic acid, and incubated on ice for the first 20 min, and finished with 10 min incubation at 37˚C. The pH of each reaction was monitored.

46 Supplemental Figures and Tables

A B Ac 5×107 Ac 5×107

4×107 Gc * 4×107 * 7 3×10 3×107 Gc 2×107 2×107 1×107 1×107 0 0 C D

5×107 5×107 4×107 4×107 3×107 3×107

7 7 2×10 Ac 2×10 Ac Kdn 7 1×107 1×10 Kdn 0 0 E F 5×107 5×107 Ac 7 7 4×10 4×10 Ac 7 7 3×10 3×10 2×107 2×107 Kdn Kdn 1×107 1×107

0 0

Supplemental Figure 1. HPLC analyses for free and CMP-sialic acid via de O-acetylation. (A) Bovine submaxillary mucin (BSM) was used as a standard for O-acetyl groups of sialic acids (Sias) and (B) the de-O-acetylation treatment. O-acetyl groups and solubilizing groups such as Neu5Gc7Ac, Neu5,7Ac2 (*) can be removed with NaOH treatment at 37˚C for 30 min. Control (C, D) and ESRD (E, F) serum samples did not show any significant peak shift (0-50 min in HPLC run) after NaOH treatment (D and F), suggesting that there was no O-acetyl form accumulation. Kdn, Neu5Ac (Ac), Neu5Gc (Gc) were annotated. Y-axis showed peak counts in HPLC run.

47 D0H0 F:\LTQ-MS data\...\Neu5Ac+Neu5Gc-4pmole 1/26/2016 2:55:04 PM Standards Standards A BD0H0 F:\LTQ-MS data\...\KDO+KDN-4pmole 1/26/2016 3:37:22 PM RT: 0.00 - 35.00 SM: 15G RT: 0.00 - 35.00 SM: 15G RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G 8.33 NL: 10.80 NL: 100 100 7.34 NL: 7.75 NL: 9.94E4 1.22E5 100 7.14E4 100 2.17E5 Neu5Gc std m/z= Neu5Ac std m/z= Kdn std m/z= Kdo std m/z= 90 90 90 90 442.00- 426.00- 385.00- 355.00- 442.20 426.20 385.20 355.30 80 MS 80 MS 80 80 MS MS Neu5Ac+ Neu5Ac+ KDO+ KDO+ 70 Neu5Gc- 70 Neu5Gc- 70 70 KDN- KDN- 4pmole 4pmole 60 60 60 4pmole 60 4pmole

50 50 50 50

40 40 40 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 30

20 20 20 2.80 20 11.78 9.26 10 10 12.24 10 8.33 10 8.72 3.09 5.87 10.14 12.79 14.66 24.27 26.86 29.94 34.78 5.07 5.56 12.90 16.28 20.63 23.65 26.44 29.16 31.88 34.06 3.60 8.97 10.73 14.75 16.62 21.01 23.59 26.75 28.23 32.51 34.30 4.91 9.80 10.74 13.89 16.57 18.87 24.47 26.92 29.72 31.69 0 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min) Time (min) Time (min)

Neu5Ac+Neu5Gc-4pmole #628-715 RT: 7.95-8.78 AV: 88 NL: 5.44E4 Neu5Ac+Neu5Gc-4pmole #899-987 RT: 10.53-11.37 AV: 89 NL: 8.01E4 KDO+KDN-4pmole #529-629 RT: 7.01-7.96 AV: 101 NL: 3.49E4 KDO+KDN-4pmole #567-692 RT: 7.37-8.56 AV: 126 NL: 8.91E4 T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] 442.17 426.17 385.14 355.13 100 5.63 e+004 100 8.23 e+004 100 3.51 e+004 100 8.99 e+004 90 90 90 90

80 80 80 80

70 70 70 70

60 60 60 60

50 50 426.41 50 50 357.32 40 40 40 40

Relative Abundance 442.32 Relative Abundance 30 30 Relative Abundance 30 Relative Abundance 30 427.17 20 443.17 20 20 20 354.24 356.13 427.40 386.14 451.34 443.32 10 10 429.39 10 386.06 10 356.30 357.36 385.32 386.39 437.33 439.94 441.41 444.17 444.87 447.09 448.94 449.98 452.34 420.42 422.35 424.33 425.18 428.40 430.39 431.12 432.89 434.17 382.31 382.98 383.34 383.93 384.26 385.00 386.96 353.56 354.08 354.36 355.09 355.25 356.02 356.75 357.14 357.81 0 0 0 0 438 440 442 444 446 448 450 452 422 424 426 428 430 432 434 382.0 382.5 383.0 383.5 384.0 384.5 385.0 385.5 386.0 386.5 387.0 353.5 354.0 354.5 355.0 355.5 356.0 356.5 357.0 357.5 358.0 m/z m/z m/z m/z Control serum ESRD serum C D0H0 F:\LTQ-MS data\...\CKD-012616\C-3 1/26/2016 5:01:53 PM D D0H0 F:\LTQ-MS data\...\CKD-012616\S-13 1/26/2016 8:33:12 PM RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 35.00 SM: 15G RT: 0.00 - 35.00 SM: 15G 2.28 NL: 3.09 NL: 31.57 NL: 10.56 NL: 100 1.09E4 100 10.50 Neu5Ac 6.61E4 100 1.29E4 100 Neu5Ac 5.11E5 m/z= m/z= m/z= m/z= 90 90 90 90 442.00- 426.00- 442.00- 426.00- 442.20 426.20 442.20 426.20 80 2.55 80 80 MS C-3 80 MS C-3 MS S-13 MS S-13 70 70 70 70

60 4.32 60 60 60 2.45 50 no Neu5Gc peak 50 50 no Neu5Gc peak 50

40 40 40 4.51 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 30 2.26 34.28 30.58 20 20 20 20

6.46 10 28.39 10 3.06 11.81 32.24 10 2.69 10 2.13 11.99 7.96 28.04 2.10 7.18 19.42 19.88 19.76 6.03 9.14 12.88 16.11 18.42 20.81 23.01 11.53 16.94 21.08 26.86 31.00 34.39 4.33 20.05 2.11 5.90 8.00 13.17 15.20 19.87 20.50 25.03 29.35 4.71 8.44 15.20 19.32 22.49 28.01 30.31 32.51 0 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min) Time (min) Time (min)

S-13 #656-747 RT: 7.94-8.78 AV: 92 NL: 1.90E4 S-13 #893-1010 RT: 10.15-11.26 AV: 118 NL: 2.96E5 C-3 #651-739 RT: 7.95-8.79 AV: 89 NL: 2.19E4 C-3 #869-1029 RT: 10.02-11.54 AV: 161 NL: 2.83E4 T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] 442.31 426.17 442.31 426.17 100 100 100 100 2.67e+005 2.83e+0.04 90 90 90 90 80 80 80 80 70 70 70 70 442.45 60 60 60 60 50 50 426.41 50 50 427.40 437.29 40 40 40 40 Relative Abundance 30 437.38 Relative Abundance 30 Relative Abundance Relative Abundance 439.18 30 443.32 30 427.66 434.00 443.32 431.24 427.17 438.95 451.34 429.38 20 20 20 447.92 20 427.17 444.08 452.09 452.17 428.40 10 438.30 10 427.40 10 10 440.00 444.20 447.14 449.27 451.34 437.96 444.42 450.94 452.87 420.42 441.41 446.05 453.13 420.42 424.26 428.40 431.24 433.99 422.31 424.32 422.31 425.26 430.26 432.24 439.02 441.41 445.14 446.95 449.82 425.33 430.39 432.24 433.10 435.00 0 0 0 0 438 440 442 444 446 448 450 452 422 424 426 428 430 432 434 438 440 442 444 446 448 450 452 422 424 426 428 430 432 434 m/z m/z m/z m/z

D0H0 F:\LTQ-MS data\...\CKD-012616\C-3 1/26/2016 5:01:53Control PM serum D0H0 F:\LTQ-MS data\...\CKD-012616\S-13 1/26/2016 8:33:12ESRD PM serum 1 E F RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 35.00 SM: 15G RT: 0.00 - 35.00 SM: 15G 2.42 2.94 NL: NL: 7.25 NL: NL: 100 1.28E4 100 4.44E4 100 Kdn 1.01E5 100 2.40 3.90E4 m/z= m/z= m/z= m/z= 90 90 90 90 385.00- 2.41 355.00- 385.00- 2.52 355.00- 385.20 355.30 385.20 355.30 80 MS C-3 80 MS C-3 80 MS S-13 80 MS S-13 70 70 70 70

60 60 60 60 3.12 4.34 2.18 Kdn no Kdo peak 15.66 50 7.25 50 50 50 3.25 40 40 3.02 40 40 Kdo

Relative Abundance Relative Abundance Relative Abundance Relative Abundance 10.74 16.08 30 4.54 30 30 34.14 30 10.49 33.49 4.23 16.38 4.40 7.64 20 20 20 20 11.14 16.82 2.45 31.68 32.33 8.71 10 10 10 2.82 8.34 10 20.11 18.88 21.14 8.38 13.34 13.04 22.72 33.59 33.88 4.78 8.53 12.15 15.00 24.37 28.18 31.12 34.39 5.27 11.97 14.81 17.48 19.33 22.68 26.88 30.10 4.70 11.27 11.55 30.91 7.01 23.49 29.68 31.64 15.91 19.38 21.65 24.47 26.84 0 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min) Time (min) Time (min)

C-3 #536-610 RT: 6.86-7.56 AV: 75 NL: 4.26E3 C-3 #590-715 RT: 7.37-8.56 AV: 126 NL: 4.29E4 S-13 #554-641 RT: 6.97-7.80 AV: 88 NL: 5.76E4 S-13 #604-659 RT: 7.45-7.97 AV: 56 NL: 3.74E4 T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] 386.89 357.32 385.14 3.51 e+004 357.32 100 100 100 100

90 90 90 90 80 3.08e+003 80 80 80 385.14 70 70 70 70 385.32 60 60 60 60

50 50 50 50

40 40 40 40 386.39 Relative Abundance 30 Relative Abundance 30 Relative Abundance 30 Relative Abundance 30 386.06 354.24 354.24 20 20 354.33 20 386.14 20 8.99 e+004 357.18 355.13 385.99 357.36 357.92 354.12 357.36 357.92 10 382.33 384.39 10 353.88 356.29 10 384.24 10 383.93 357.17 382.33 385.32 353.88 356.29 382.59 383.09 383.70 384.79 385.40 353.57 353.99 354.36 355.04 355.25 355.78 356.04 356.73 356.92 357.61 382.92 383.26 384.11 384.39 385.07 386.06 386.39 386.89 354.33 355.08 355.24 356.11 356.92 0 0 0 0 382.0 382.5 383.0 383.5 384.0 384.5 385.0 385.5 386.0 386.5 387.0 353.5 354.0 354.5 355.0 355.5 356.0 356.5 357.0 357.5 358.0 382.0 382.5 383.0 383.5 384.0 384.5 385.0 385.5 386.0 386.5 387.0 353.5 354.0 354.5 355.0 355.5 356.0 356.5 357.0 357.5 358.0 m/z m/z m/z m/z

D0H0 F:\LTQ-MS data\...\CKD-012616\S-7 1/26/2016 7:50:56ESRD PM serum 2 D0H0 F:\LTQ-MS data\...\CKD-012616\S-7 1/26/2016 7:50:56 PMESRD serum 2 G H RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G RT: 0.00 - 34.99 SM: 15G 7.25 NL: 2.45 NL: 7.25 NL: 2.45 NL: 100 Kdn 1.08E5 100 4.03E4 100 1.08E5 100 4.03E4 m/z= m/z= m/z= m/z= 90 90 90 90 385.00- 355.00- 385.00- 355.00- 385.20 355.30 8.27 sec 385.20 355.30 80 80 80 80 10.77 sec MS S-7 MS S-7 MS S-7 MS S-7 70 70 Kdo 70 RTAc =0.7831 70 RTAc =1.024 60 60 4.60 60 60 4.60 8.27 27.97 15.68 8.27 27.97 15.68 50 50 50 50 7.68 15.98 7.68 15.98 19.48 19.48 40 40 2.15 40 40 2.15 14.99 19.75 14.99 19.75 Relative Abundance Relative Abundance 16.26 Relative Abundance Relative Abundance 16.26 30 30 30 30 10.77 16.48 20.05 10.77 16.48 20.05 26.25 11.95 26.25 11.95 20 14.94 20 19.02 20 14.94 20 19.02 2.49 2.99 31.47 32.45 8.29 2.49 2.99 31.47 32.45 8.29 10 9.03 10 7.09 10 9.03 10 7.09 13.73 15.74 21.13 24.93 27.85 31.34 13.73 15.74 21.13 24.93 27.85 31.34 2.23 17.18 24.19 24.59 2.23 17.18 24.19 24.59 0 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min) Time (min) Time (min)

S-7 #560-635 RT: 7.03-7.74 AV: 76 NL: 6.75E4 S-7 #604-666 RT: 7.45-8.03 AV: 63 NL: 3.90E4 S-7 #653-703 RT: 7.91-8.38 AV: 51 NL: 3.83E4 S-7 #925-987 RT: 10.43-11.02 AV: 63 NL: 4.00E4 T: FTMS + p ESI Full ms [200.00-1000.00] 6.90e+004 T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] T: FTMS + p ESI Full ms [200.00-1000.00] 385.14 357.31 385.16 357.31 100 100 100 100

90 90 90 Mass 385.16 90 382.13 80 80 80 3.93e+004 80 70 70 70 70 385.16 60 60 60 60

50 50 50 50 40 40 1.21e+004 40 40 Mass 355.17

Relative Abundance Relative Abundance 355.13 Relative Abundance Relative Abundance 30 30 30 30

354.24 354.24 6.29e+003 20 386.14 20 20 386.17 20 355.17 356.09 357.92 354.12 357.36 357.36 384.24 357.18 354.06 10 10 356.28 357.92 10 383.13 385.32 386.08 10 356.29 357.08 382.33 386.39 353.88 354.33 382.21 354.33 383.09 383.80 384.11 384.39 384.97 385.36 386.06 386.89 355.08 355.38 355.83 357.14 382.56 383.26 384.16 384.39 385.08 386.02 386.39 353.88 355.08 355.24 356.20 356.72 356.92 357.86 0 0 0 0 382.0 382.5 383.0 383.5 384.0 384.5 385.0 385.5 386.0 386.5 387.0 353.5 354.0 354.5 355.0 355.5 356.0 356.5 357.0 357.5 358.0 382.0 382.5 383.0 383.5 384.0 384.5 385.0 385.5 386.0 386.5 387.0 353.5 354.0 354.5 355.0 355.5 356.0 356.5 357.0 357.5 358.0 m/z m/z m/z m/z Supplemental Figure 2. LC/MS analyses for free α-ketoacids including sialic acids after DMB-tagging. Peak analyses of standards (std) for (A) Neu5Gc and Neu5Ac, (B) Kdn and Kdo in LC-MS. Neu5Gc and Neu5Ac peaks in (C) control and, (D) end-stage of renal disease (ESRD) patient

48 serum. Kdn and Kdo peaks in (E) control and, (F), (G) ESRD. (H) Examples of unknown peaks that were found or significantly accumulated only in ESRD (red arrows). The same data are presented for (G) and (H) with different regions highlighted.

49 A B C 1000 1000 1000

800 800 800

600 600 600

400 400 400

200 200 200

0 0 0 D E F 100 200 200

80 150 150 60 100 100 40 50 50 20 0 0 0

Supplemental Figure 3. HPAEC-PAD Analyses for Mannose-6P. HPAEC-PAD peaks for (A) 0.2 µg (0.8 nmol) Man-6P standard for serum analysis; (B) without and (C) with external 0.2 µg Man-6P adding to the serum sample 1 hour after mannose ingestion when the serum mannose peak comes (Figure 3A). (D) Peaks for 0.2 µg Man-6P standard for urine analysis (E) without and (F) with external 0.2 µg Man-6P addition to the urine collected 4 hours after mannose ingestion when urine mannose peak comes. Man-6P is detected only in the spiked urine sample (arrow) (F), suggesting that Man-6P could not exist in human serum and urine, as well. Y-axis showed peak counts in HPAEC-PAD run.

50 A Free Sias B CMP - Kdn C CMP-Neu5Ac 100 pmole Stds 100 pmole Stds 100 pmole stds 100 pmole Stds 100 pmole stds 100 pmole stds 100 pmole Stds 100 pmole CMP Kdn 100 pmole CMP Neu5Ac Kdn Kdn Kdn Kdn3×107 3×107 3×107 7 Kdn 7 7 3×107 Ac 3×310×107 Ac 3×3×1010 Ac 3×10 Ac Ac Ac Gc Gc Gc 2×107 2×107 2×107

Gc (counts) (counts) 7 (counts) Gc 7 7 22××10107 2×210×107 2×2×1010 (counts) 7 (counts) 7 7 (counts) 1×10 Kdn1×10 1×10

7 77 7 HPLC Peak 11××10107 1×110×10 HPLC Peak 1×1×1010 HPLC Peak 0 0 0 HPLC Peak HPLC Peak HPLC Peak HPLC Peak (counts) 00 HPLC Peak (counts) 00 HPLC Peak (counts) 0

D Free Sias plus NaBH4 E CMP-Kdn plus NaBH4 F CMP-Neu5Ac plus NaBH4 100 pmole Stds 100 pmole Stds 100 pmole Stds 100 100pmole pmole Stds stds plus NaBH4 100 pmole100 pmole CMP stds Kdn plus NaBH4 100 pmole stds Kdn Kdn 100 pmole CMP-Ac plus NaBH4 3×107 7 Kdn 3×10 3×107 3×107 Ac 7 Ac 3×10 33××1010 Ac 3×310×1077 Ac Gc Gc 2×107 7 Gc 2×10 2×107 (counts) 7 (counts) 7 7 (counts) 2×2×1010 22××1010 2×210×1077

(counts) 7 1×10 7 7 (counts) 1×10 1×10 7

HPLC Peak 7 1×10 HPLC Peak 77 1×10 11××1010 Kdn 1×110×10 HPLC Peak 0 0 0 HPLC Peak HPLC Peak (counts) HPLC Peak (counts) 0 HPLC Peak 0 0 HPLC Peak (counts) 00

Supplemental Figure 4. DMB-HPLC analyses for free and CMP-activated form of sialic acids. DMB-HPLC peaks for (A) free Sia 100 pmoles standards (Kdn, Neu5Gc (Gc) and Neu5Ac (Ac)); (B) CMP-Kdn; (C) CMP-Neu5Ac. (D) Free form of Kdn, Neu5Gc and Neu5Ac are sensitive to

NaBH4 treatment, but (E) CMP-Kdn and (F) CMP-Neu5Ac are resistant to NaBH4. Y-axis showed peak counts in HPLC run.

51 aa (hNANS) 39 40 41 42 43 44 45 Nucleotide no.

Species 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 H. sapiens (HUMAN) A T G A T C C G C A T G G C C A A G G A G P. troglodytes (CHIMPANZEE) A T G A T C C G C A T G G C C A A G G A G P. paniscus (BONOBO) A T G A T C C G C A T G G C C A A G G A G P. abelii (SUMATRAN ORANGUTAN) A T G A T C C G C A T G G C C A A G G A G M. mulatta (RHESUS MONKEY) A T G A T C C G C A T G G C C A A G G A G C. c. imitator (COLOMBIAN WHITE-FACED CAPUCHIN) A T G A T C C G C G T G G C C A A G G A G C. jacchus (COMMON MARMOSET) A T G A T C C G C G T G G C C A A G G A G C. ferus (BACTRIAN CAMEL) A T G A T C C G C A C C G C C A A G G A G E. caballus (HORSE) A T G A T C C G C A C G G C C A A G G A G M. murinus (GRAY MOUSE LEMUR) A T G A T C C G C A C C G C C A A G G A G S. scrofa (WILD BOAR) A T G A T C C G T A T G G C C A A G G A G N. a. asiaeorientalis (NARROW-RIDGED FINLESS PORPOISE) A T G A T C C G C A T G G C C A A G G A G O. orca (ORCA) A T G A T C C G C A T G G C C A A G G A G T. truncatus (COMMON BOTTLENOSE DOLPHIN) A T G A T C C G C A T G G C C A A G G A G L. vexillifer (BAJI-DOPHIN) A T G A T C C G C A T G G C C A A G G A G P. catodon (SPERM WHALE) A T G A T C C G C A T G G C C A A G G A G E. asinus (DONKEY) A T G A T C C G C A C G G C C A A G G A G A.jubatus (CHEETAH) A T G A T C C G C A C G G C C A A G G A G U. a. horribilis (GRIZZLY BEAR) A T G A T C C G C A C G G C C A A G G A G B. bubalis (BUFFALO) A T G A T C C G C A T G G C T A A G G A G Z. californianus (CALIFORNIA SEA LION) A T G A T C C G C A C G G C C A A G G A G P. t. altaica (SIBErIAN TIGER) A T G A T C C G C A C G G C C A A G G A G O. r. divergens (WALRUS) A T G A T C C G C A C G G C C A A G G A G B. b. bison (PLAINS BISON) A T G A T C C G C A T G G C T A A G G A G C. l. dingo (DINGO) A T G A T C C G C A C G G C T A A G G A G C. l. familiaris (DOMESTIC DOG) A T G A T C C G C A C G G C T A A G G A G A. melanoleuca (GIANT PANDA) A T G A T C C G C A C G G C T A A G G A G B. mutus (WILD YAK) A T G A T C C G C A T G G C T A A G G A G V. vulpes (RED-FOX) A T G A T C C G C A C G G C T A A G G A G C. hircus (GOAT) A T G A T C C G C A T G G C T A A G G A G B. taurus (CATTLE) A T G A T C C G C A T G G C T A A G G A G C. canadensis (NORTH AMERICAN BEAVER) A T G A T C C G C A C G G C C A A G G A G O. aries (SHEEP) A T G A T C C T C A T G G C T A A G G A G C. s. simum (SOUTHERN WHITE RHINOZEROS) A T G A T C C G C A C G G C C A A G G A G O. v. texanus (WHITE-TAILED DEER) A T G A T C C G C A T G G C T A A G G A G L. africana (AFRICAN BUSH ELEFANT) A T G A T C C G C A C G G C G A A G G A G I. tridecemlineatus (THIRTEEN-LINED GROUND SQUIRREL) A T G A T C C G C A C A G C C A A G G A G C. griseus (CHINESE HAMSTER) A T G A T C C G C A C T G C C A A G G A G M. musculus (HOUSE MOUSE) A T G A T C C G C A C T G C C A A G G A G G. japonicus (SCHLEGELS JAPANESE GECKO) A T G A T T C G C G T G G C C A A G G A G P. muralis (COMMON WALL LIZARD) A T G A T C C G A G T G G C T A A G G A A N. scutatus (TIGER SNAKE) A T G A T C C G G G T G G C T A A G G A A N. meleagris (HELMETED GUINEAFOWL) A T G A T C C G C A T G G C C A A G G A C G. gallus (RED JUNGLEFOWL) A T G A T C C G C A T G G C C A A G G A G N. perdicaria (CHILEAN TINAMOU) A T G A T C C G C A T G G C C A A G G A G A. cunicularia (BURROWING OWL) A T G A T C C G C A T G G C C A A G G A C D. novaehollandiae (EMU) A T G A T C C G C A T G G C C A A G G A T C. mydas (GREEN SEA TURTLE) A T G A T C C G C A T G G T C A A G G A G E. telfairi (LESSER HEDGEHOG TENREC) A T G A T C C G C A C G G C G A A G G A G I. punctatus (CHANNEL CATFISH) A T G A T C C G A A T G G C C A A A G A T C. porosus (SALTWATER CROCODILE) A T G A T C C G C A T G G C C A A G G A G D. rerio (ZEBRAFISH) A T G A T C A A A A T G G C A A A G G A C C. adamanteus (EASTERN DIAMONDBACK RATTLESNAKE) A T G A T C C G G C T G G T C A A G G A C X. tropicalis (WESTERN CLAWED FROG) A T G A T C C G C A T G G C A A A G G A C O. mykiss (RAINBOW TROUT) A T G A T C A A G A T G G C C A A G G A C N. parkeri (HIGH HIMALAYA FROG) A T G A T T C G C A T G G C T A A G G A C T. c. triunguis (THREE-TOED BOX TURTLE) A T G A T C C G C A T G G T C A A G G A G S. salar (ATLANTIC SALMON) A T G A T C A A G A T G G C C A A G G A C O. anatinus (PLATYPUS) A T G A T C C G C A T G G T C A A G G A T P. cinereus (KOALA) A T G A T C C G G A T G G C A A A G G A G V. ursinus (COMMON WOMBAT) A T G A T C C G G A T G G C A A A G G A G M. domestica (GRAY SHORT-TAILED OPOSSUM) A T G A T C C G G A T G G C C A A G G A G

Actinopterygii T42 Amphibia V42 Aves M42 Mammalia L42 Reptilia

Supplemental Figure 5. Part of a multiple alignment of NANS nucleotide sequences Codons specifying Methionine (M) and Leucine (L) at position 42 in human NANS (giving rise to NANS with Kdn synthase activity), are shaded in light and dark blue, respectively, while Threonine (T) and Valine (V) are shaded in light and dark grey. Species are shaded according to

52 classes: Actinopterygii (light purple), Amphibia (pink), Aves (dark purple) Mammalia (green), Reptilia (blue).

53 Supplemental Table 1. Standards used for Mass Spectrometry Analysis.

α-ketoacids Abbreviation Mass Source 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid Kdn 385.14 Commercial 3-deoxy-D-manno-oct-2-ulosonic acid Kdo 355.13 Commercial N-glycolylneuraminic acid Neu5Gc 442.16 Commercial 5,7-diamino-3,5,7, 9-tetradeoxy-D-glycero-D- Leg 473 Commercial galacto-non-2-ulosonic (legionaminic) acid 7-O-acetyl-N-glycolylneuraminic acid Neu5Gc7Ac 484.17 *BSM N-acetylneuraminic acid Neu5Ac 426.17 Commercial 8-O-acetyl-N-glyconeuraminic acid Neu5Gc8Ac 484.17 BSM 5,7- diamino-3,5,7,9-tetradeoxy-L-glycero-L- Pse 451 Commercial manno-non-2-ulosonic (pseudaminic) acid 7-O-acetyl-N-acetylneuraminic acid Neu5,7Ac2 468.18 BSM 9-O-acetyl-N-glyconeuraminic acid Neu5Gc9Ac 484.17 BSM 8-O-acetyl-N-acetylneuraminic acid Neu5,8Ac2 468.18 BSM 9-O-acetyl-N-acetylneuraminic acid Neu5,9Ac2 468.18 BSM 8,9-di-O-acetyl-N-acetylneuraminic acid Neu5,8,9Ac3 510 BSM 7,9-di-O-acetyl-N-acetylneuraminic acid Neu5,7,9Ac3 510 BSM

Each standard for α-ketoacids including sialic acids is available in commercial inventories including *bovine submaxillary mucin (BSM).

54 Supplemental Table 2. Kdn relative to Neu5Ac increased in various mammalian cells after mannose feeding.

Table 2-1. Kdn relative to Neu5Ac in cytosol.

Mannose concentration Cell line 0 mM 1 mM 3 mM 5 mM 10 mM 15 mM HEK293A 0.24 0.27 0.31 0.31 0.38 0.42 HUVEC 0.07 0.22 0.22 1.01 3.82 3.02 BJAB K88 1.47 2.19 3.66 4.87 8.84 14.23 BJAB K20 5.18 10.39 49.12 28.84 26.91 90.62

Table 2-2. Kdn relative to Neu5Ac in growth media.

Mannose concentration Cell line 0 mM 5 mM 15 mM HEK293A 0.127 0.526 1.418 HUVEC 1.128 1.130 1.624 BJAB K88 0.121 0.125 0.239 BJAB K20 0.149 0.453 1.780 PMI WT 0.098 0.416 0.604 PMI KO# 0.527 1.502 1.181 HK2 0.815 3.386 5.085

#The corresponding amounts of mannose for PMI KO cells were 25 µM, 250 µM and 1 mM.

Table 2-3. *Total conjugated Kdn relative to Neu5Ac in total cell lysate with NaOH and NaBH4.

Mannose concentration Cell line 0 mM 5 mM 15 mM HEK293A 0.000 0.000 0.000 HUVEC 0.001 0.002 0.003 BJAB K88 0.002 0.011 0.024 BJAB K20 0.013 0.054 0.165 PMI WT 0.000 0.003 0.004 PMI KO# 0.003 0.004 0.003

*Free sialic acids (Sias) and CMP-Sias were destroyed by NaBH4 treatment. It showed total cell conjugated form of Sias. #The corresponding amounts of mannose for PMI KO cells were 25 µM, 250 µM and 1 mM.

55 Table 2-4. Kdn relative to Neu5Ac in membrane fraction.

Mannose concentration Cell line 0 mM 15 mM HEK293A 0 0.0020 BJAB K88 0 0.064 BJAB K20 0.0042 0.11 PMI WT 0 0.0014 PMI KO# 0.012 0.0005 HK2 0 0.0035 #The corresponding amounts of mannose for PMI KO cells were 25 µM and 1 mM.

Table 2-5. Kdn relative to Neu5Ac in membrane fraction of HEK293A with fish Cmas transfection.

Mannose concentration vector 0 mM 15 mM non-transfected 0 0.0014 empty vector 0 0.0019 *Cmas1 0.0043 0.0163 *Cmas2 0.0076 0.0323

*Two types of zebrafish CMP-sialic acid synthetases (Cmas) with differential substrate affinity were transiently expressed in HEK293A cells, and non-transfected and empty vector transfected cells were set as controls. Cells were harvested 36 hours post mannose feeding (0 or 15 mM) and membrane fractions were prepared to analyze glycosidically-conjugated form of sialic acids by DMB-HPLC analysis (Kdn relative to Neu5Ac).

56 Supplemental Table 3. Identity and homology of human and other vertebrate NANS proteins.

Accession number Species Identity Homology Class (Protein) Acinonyx jubatus XP_014934957.1 96% 98% Mammalia Ailuropoda XP_002914976.1 96% 98% Mammalia melanoleuca Athene cunicularia XP_026721911.1 84% 92% Aves Bison bison bison XP_010845354.1 97% 99% Mammalia Bos mutus XP_005906043.1 97% 99% Mammalia Bos taurus NP_001039947.1 97% 99% Mammalia Bubalus bubalis XP_006063515.1 97% 99% Mammalia Callithrix jacchus XP_008998209.1 98% 99% Mammalia Camelus ferus XP_006187837.1 97% 98% Mammalia Canis lupus dingo XP_025287783.1 96% 98% Mammalia Canis lupus XP_538746.2 96% 98% Mammalia familiaris Capra hircus XP_005683962.2 97% 99% Mammalia Castor canadensis XP_020036388.1 97% 98% Mammalia Cebus capucinus XP_017383327.1 98% 99% Mammalia imitator Ceratotherium simum XP_004423294.1 96% 98% Mammalia simum Chelonia mydas XP_007066359.1 85% 93% Reptilia Cricetulus griseus XP_003500141.1 95% 98% Mammalia Crocodylus porosu XP_019410688.1 88% 93% Reptilia Crotalus AFJ51454.1 84% 94% Reptilia adamanteus Danio rerio NP_996660.1 79% 92% Actinopterygii Dromaius XP_025955160.1 85% 94% Aves novaehollandiae Echinops telfairi XP_004711046.1 92% 97% Mammalia Equus asinus XP_014715223.1 96% 98% Mammalia Equus caballus XP_023485493.1 96% 98% Mammalia Gallus gallus NP_001007976.1 87% 94% Aves Gekko japonicus XP_015281630.1 84% 94% Reptilia Homo sapiens NP_061819.2 100% 100% Mammalia Ictalurus punctatus NP_001187692.1 78% 91% Actinopterygii Ictidomys XP_005326366.1 96% 98% Mammalia tridecemlineatus Lipotes vexillifer XP_007461720.1 97% 98% Mammalia Loxodonta africana XP_003407624.1 95% 98% Mammalia Macaca mulatta EHH23940.1 99% 99% Mammalia

57 Microcebus XP_012624488.1 97% 99% Mammalia murinus Monodelphis domestica XP_001364128.1 87% 94% Mammalia (Methateria) Mus musculus NP_444409.1 95% 97% Mammalia Nanorana parkeri XP_018407945.1 76% 89% Amphibia Neophocaena asiaeorientalis XP_024590225.1 97% 98% Mammalia asiaeorientalis Notechis scutatus XP_026528761.1 84% 94% Reptilia Nothoprocta XP_025893877.1 84% 91% Aves perdicaria Numida meleagris XP_021236216.1 87% 94% Aves Odobenus rosmar XP_004392385.1 95% 98% Mammalia divergens Odocoileus XP_020761234.1 96% 99% Mammalia virginianus texanus Oncorhynchus XP_021429201.1 79% 91% Actinopterygii mykiss Orcinus orca XP_004271519.1 96% 98% Mammalia Ornithorhynchus XP_001506765.1 89% 95% Mammalia (Monotremata) anatinus Ovis aries XP_004005320.3 97% 99% Mammalia Pan paniscus XP_008972309.1 99% 99% Mammalia Pan troglodytes XP_024201562.1 100% 100% Mammalia Panthera tigris XP_007089351.1 96% 98% Mammalia altaica Phascolarctos XP_020860268.1 90% 94% Mammalia (Methateria) cinereus Physeter catodon XP_023982297.1 97% 98% Mammalia Podarcis muralis XP_028568080.1 85% 94% Reptilia Pongo abelii XP_002820068.1 99% 99% Mammalia Salmo salar ACI33492.1 79% 91% Actinopterygii Sus scrofa NP_001172068.1 97% 99% Mammalia Terrapene carolina XP_024062240.2 85% 93% Reptilia triunguis Tursiops truncatus XP_004322851.1 97% 98% Mammalia Ursus arctos XP_026361945.1 95% 98% Mammalia horribilis Vombatus ursinus XP_027714871.1 89% 94% Mammalia (Methateria) Vulpes vulpes XP_025868953.1 96% 98% Mammalia Xenopus tropicalis XP_002935918.1 76% 90% Amphibia Zalophus XP_027470724.1 95% 98% Mammalia californianus

Identity (%) and homology (%) of vertebrate NANS protein sequences to H. sapiens NANS

58 NCBI Reference Sequence Numbers are given and species are shaded according to classes: Actinopterygii, Amphibia, Aves, Mammalia, Reptilia.

59 Supplementary Table 4. Eukaryotic NANS sequences

Accession Accession Common Species Class Order Family number number name (Protein) (mRNA) Acinonyx XP_0149349 XM_0150794 Mammalia Carnivora Felidae Cheetah jubatus 57.1 71.2 Ailuropoda XP_0029149 XM_0029149 Giant Mammalia Carnivora Ursidae melanoleuca 76.1 30.3 panda Athene XP_0267219 XM_0268661 Burrowin Aves Strigiformes Strigidae cunicularia 11.1 10.1 g owl Bison bison XP_0108453 XM_0108470 Plains Mammalia Artiodactyla Bovidae bison 54.1 52.1 bison XP_0059060 XM_0059059 Bos mutus Mammalia Artiodactyla Bovidae Wild yak 43.1 81.2 NP_0010399 NM_0010464 Bos taurus Mammalia Artiodactyla Bovidae Cattle 47.1 82.1 Bubalus XP_0060635 XM_0060634 Mammalia Artiodactyla Bovidae Buffalo bubalis 15.1 53.2 Callithrix XP_0089982 XM_0089999 Common Mammalia Primates Callitrichidae jacchus 09.1 61.1 marmoset Camelus XP_0061878 XM_0061877 Bactrian Mammalia Artiodactyla Camelidae ferus 37.1 75.2 Camel Canis lupus XP_0252877 XM_0254319 Mammalia Carnivora Canidae Dingo dingo 83.1 98.1 Canis lupus Domestic Mammalia Carnivora Canidae XP_538746.2 XM_538746.6 familiaris dog XP_0056839 XM_0056839 Capra hircus Mammalia Artiodactyla Bovidae Goat 62.2 05.3 North Castor XP_0200363 XM_0201807 Mammalia Rodentia Castoridae American canadensis 88.1 99.1 beaver Colombia Cebus XP_0173833 XM_0175278 n white- capucinus Mammalia Primates Cebidae 27.1 38.1 faced imitator capuchin Ceratotheriu Southern Perissodactyl Rhinocerotida XP_0044232 XM_0044232 m simum Mammalia white a e 94.1 37.2 simum rhinozeros Chelonia XP_0070663 XM_0070662 Green sea Reptilia Testudines Cheloniidae mydas 59.1 97.2 turtle Cricetulus XP_0035001 XM_0274031 Chinese Mammalia Rodentia Cricetidae griseus 41.1 45.1 hamster Crocodylus XP_0194106 XM_0195551 Saltwater Reptilia Crocodilia Crocodylidae porosus 88.1 43.1 crocodile Eastern diamondb Crotalus Reptilia Squamata Viperidae AFJ51454.1 JU175930.1 ack adamanteus rattlesnak e

60 Actinopter Cypriniform Danio rerio Cyprinidae NP_996660.1 NM_206829.1 Zebrafish ygii es Dromaius Casuariiform XP_0259551 XM_0260993 novohollandi Aves Casuariidae Emu es 60.1 75.1 ae Lesser Echinops XP_0047110 XM_0047109 Mammalia Afrosoricida Tenrecidae hedgehog telfairi 46.1 89.1 tenrec Perissodactyl XP_0147152 XM_0148597 Equus asinus Mammalia Equidae Donkey a 23.1 37.1 Equus Perissodactyl XP_0234854 XM_0236297 Mammalia Equidae Horse caballus a 93.1 25.1 Red NP_0010079 NM_0010079 Gallus gallus Aves Galliformes Phasianidae junglefow 76.1 75.1 l Schlegel's Gekko XP_0152816 XM_0154261 Reptilia Squamata Gekkonidae Japanese japonicus 30.1 44. gecko Homo Mammalia Primates Hominidae NP_061819.2 NM_018946.4 Human sapiens Ictalurus Actinopter NP_0011876 NM_0012007 Channel Siluriformes Ictaluridae punctatus ygii 92.1 63.2 catfish Thirteen- Ictidomys XP_0053263 XM_0053263 lined tridecemline Mammalia Rodentia Sciuridae 66.1 09.2 ground atus squirrel Lipotes XP_0074617 XM_0074616 Baji- Mammalia Artiodactyla Lipotidae vexillifer 20.1 58.1 dolphin African Loxodonta XP_0034076 XM_0034075 Mammalia Proboscidea Elephantidae bush africana 24.1 [ 76.2 elephant Macaca Cercopithecid XM_0151172 Rhesus Mammalia Primates EHH23940.1 mulatta ae 29.2 macaque Gray Microcebus Cheirogaleida XP_0126244 XM_0127690 Mammalia Primates mouse murinus e 88.1 34.1 lemur Gray Monodelphis Didelphimor XP_0013641 XM_0013640 short- Mammalia Didelphidae domestica phia 28.1 91.4 tailed opossum Mus House Mammalia Rodentia Muridae NP_444409.1 NM_053179.3 musculus mouse High Nanorana XP_0184079 XM_0185524 Amphibia Anura Dicroglossidae Himalaya parkeri 45.1 43.1 frog Neophocaen a Narrow- asiaeoriental XP_0245902 XM_0247344 ridged Mammalia Artiodactyla Phocoenidae is 25.1 57. finless asiaeoriental porpoise is

61 Notechis XP_0265287 XM_0266729 Tiger Reptilia Squamata Elapidae scutatus 61.1 76.1 snake Nothoprocta Tinamiforme XP_0258938 XM_0260380 Chilean Aves Tinamidae perdicaria s 77.1 92. tinamou Helmeted Numida XP_0212362 XM_0213805 Aves Galliformes Numididae guineafow melagris 16.1 41.1 l Odobenus XP_0043923 XM_0043923 rosmarus Mammalia Carnivora Odobenidae Walrus 85.1 28.1 divergens Odocoileus XP_0207612 XM_0209055 White- virginianus Mammalia Artiodactyla Cervidae 34.1 75.1 tailed deer texanus Oncorhynch Actinopter Salmoniform XP_0214292 GBTD011617 Rainbow Salmonidae us mykiss ygii es 01.1 35.1 trout XP_0042715 XM_0042714 Orcinus orca Mammalia Artiodactyla Delphinidae Orca 19.1 71.1 Ornithorhync Ornithorhynch XP_0015067 XM_0015067 Mammalia Monotremata Platypus hus anatinus idae 65 15.5 XP_0040053 XM_0040052 Ovis aries Mammalia Artiodactyla Bovidae Sheep 20.3 71.3 XP_0089723 XM_0089740 Pan paniscus Mammalia Primates Hominidae Bonobo 09.1 61.2 Pan XP_0242015 XM_0243457 Chimpanz Mammalia Primates Hominidae troglodytes 62.1 94.1 ee Panthera XP_0070893 XM_0070892 Siberian Mammalia Carnivora Felidae tigris altaica 51.1 89.2 tiger Phasolarctos Diprotodonti Phascolarctida XP_0208602 XM_0210046 Mammalia Koala cinereus a e 68.1 09.1 Physeter XP_0239822 XM_0241265 Sperm Mammalia Artiodactyla Physeteridae catodon 97.1 29.2 whale Podarcis XP_0285680 XM_0287122 Common Reptilia Squamata Lacertidae muralis 80.1 47.1 wall lizard XP_0028200 XM_0028200 Sumatran Pongo abelii Mammalia Primates Hominidae 68.1 22.4 orangutan Actinopter Salmoniform Atlantic Salmo salar Salmonidae ACI33492.1 BT045230.1 ygii es salmon NP_0011720 NM_0011851 Sus scrofa Mammalia Artiodactyla Suidae Wild boar 68.1 39.1 Terrapene Three- XP_0240622 XM_0266463 carolina Reptilia Testudines Emydidae toed box 40.2 79.2 triunguis turtle Common Tursiops XP_0043228 XM_0043228 Mammalia Artiodactyla Delphinidae bottlenose truncatus 51.1 03.2 dolphin Ursus arctos XP_0263619 XM_0265061 Grizzly Mammalia Carnivora Ursidae horribilis 45.1 60.1 bear Vulpes XP_0258689 XM_0260131 Mammalia Carnivora Canidae Red-fox vulpes 53.1 68.1 Vombatus Diprotodonti XP_0277148 XM_0278590 Common Mammalia Vombatidae ursinus a 71.1 70.1 wombat

62 Western Xenopus XP_0029359 XM_0029358 Amphibia Anura Pipidae clawed tropicalis 18.1 72.4 frog Zalophus XP_0274707 XM_0276149 California Mammalia Carnivora Otariidae californianus 24.1 23. sea lion

Vertebrate NANS sequences: Taxonomy, common names and accession numbers Taxonomy and common names of vertebrates included in phylogenetic analyses (Figure 5) are shown with species shaded according to classes: Actinopterygii (light purple), Amphibia (pink), Aves (dark purple) Mammalia (green), Reptilia (blue). NCBI Reference Sequence Numbers of NANS protein and mRNA sequences.

63