Enzyme Of Sialic Acid Biosynthesis Research Articles

P.3.4 In vivo GNE interactions

GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. We have previously shown that GNE interacts with alpha-actinin1 and alpha-actinin2 in vitro, either at different interaction sites on the GNE protein, or alternatively by changing the conformational state of GNE. In the current studies we have analyzed these interactions by the bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells and now demonstrate these interactions also in vivo. In addition, to elucidate the interaction domains in GNE, we have dissected the GNE molecule, first in its 2 main domains, epimerase and kinase. We have generated those TAG proteins in the insect cells/baculovirus system and performed pull down and immunoprecipitation assays, also in the presence of both alpha-actinins. Furthermore, interactions of these “half domain” proteins were also analyzed in living cells by the BiFC methodology. The potential of frequent mutations in GNE myopathy, such as the Middle Eastern founder mutation M712T, to disrupt such interactions will be shown. These studies could contribute new understandings on the mechanism of GNE in health and disease. GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. We have previously shown that GNE interacts with alpha-actinin1 and alpha-actinin2 in vitro, either at different interaction sites on the GNE protein, or alternatively by changing the conformational state of GNE. In the current studies we have analyzed these interactions by the bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells and now demonstrate these interactions also in vivo. In addition, to elucidate the interaction domains in GNE, we have dissected the GNE molecule, first in its 2 main domains, epimerase and kinase. We have generated those TAG proteins in the insect cells/baculovirus system and performed pull down and immunoprecipitation assays, also in the presence of both alpha-actinins. Furthermore, interactions of these “half domain” proteins were also analyzed in living cells by the BiFC methodology. The potential of frequent mutations in GNE myopathy, such as the Middle Eastern founder mutation M712T, to disrupt such interactions will be shown. These studies could contribute new understandings on the mechanism of GNE in health and disease.

T.P.9 Sialyllactose reversed myopathic phenotype in symptomatic GNE myopathy model mice

GNE myopathy (GM) is caused by mutations in GNE gene, encoding an essential enzyme in sialic acid biosynthesis. We previously provided evidence that sialic acid given to clinically asymptomatic GM mice prevented the development of the myopathic phenotype, indicating that hyposialylation is one of key factors in the pathomechanism underlying GM. In this study, we examined the effect of sialic acid administration on muscle atrophy and weakness in symptomatic, older GM mice to mimic the stage of disease in symptomatic GM patients, using free sialic acid (NeuAc) and the less-metabolized sialic acid conjugate, 6′-sialyllactose (6′SL). Fifty-week-old GM mice and littermates were given either water, NeuAc, or 6’SL for about 30weeks. Voluntary exercise on running wheel was evaluated longitudinally at 10week interval, while the size, force generation, and pathology of gastrocnemius muscle (GC) were evaluated at the end of the study. Voluntary exercise in non-treated model mice markedly decreased with aging, but was maintained in 6′SL group, including one mouse that showed marked recovery to non-affected control level. The NeuAc group, on the other hand, exhibited marked decrease in voluntary wheel running similar to non-treated mice. The response in running performance correlated with findings in analysis of GC, i.e., the GC size and force production in 6′SL group was recovered to levels measured in control littermates, while those in the NeuAc group responded poorly and showed similar findings with non-treated GM mice. We show that 6′SL can ameliorate muscle atrophy and weakness in symptomatic GM mice, providing a proof of principle in the use of this compound in the clinical trial of GM patients with progressive or advanced stage of disease.

G.P.28 GNE interactions and interactors

GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. To date, over 60 different mutations in GNE have been reported to cause the disease worldwide, but a single homozygous missense mutation M712T, is common in the Middle East. The mechanisms leading from GNE mutations to the disease are not yet understood. In an attempt to elucidate the pathophysiological pathway of the disease, we have searched for proteins potentially interacting with GNE. We have previously determined that GNE interacts with α-actinin1, one of the 4 α-actinins isoforms, a family of proteins binding the actin filaments. We studied the interactions of recombinant human GNE protein with other members of the actinin family, in particular α-actinin2 which is located at the Z-disk of the muscle fiber. Using a Surface Plasmon Resonance (SPR)-Biosensor based assay, we found that GNE, in its wild type and M712T mutated forms, also binds α-actinin 2. Furthermore, GNE binds α-actinin1 and α-actinin 2 differently, either at different interaction sites on the GNE protein, or alternatively, by changing the conformational state of GNE. GNE exists in different oligomeric forms, dimers, tetramers and hexamers, which are in a dynamic interplay in the cell. To evaluate the potential effect of common mutations found in GNE myopathy patients, on the oligomeric state of GNE protein, we have investigated GNE–GNE interactions and affinity, and found the highest affinity between wtGNE and wtGNE, the lowest between mutGNE–mutGNE, and intermediate affinity between wt and mutated GNE proteins. These differences could have a direct effect on both the oligomeric state of GNE and on the dynamic interplay between the oligomeric forms of the protein. These studies could contribute new understandings on the mechanism of GNE in health and disease.

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INCORPORATION OF N-GLYCOLYLHEXOSAMINES INTO MAMMALIAN GLYCANS BY FEEDING N-GLYCOLYLGALACTOSAMINE

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern

Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells presented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.

The Gne M712T Mouse as a Model for Human Glomerulopathy

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.

Glycoprotein Hyposialylation Gives Rise to a Nephrotic-Like Syndrome That Is Prevented by Sialic Acid Administration in GNE V572L Point-Mutant Mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation.

Serum Neural Cell Adhesion Molecule Is Hyposialylated in Hereditary Inclusion Body Myopathy

Hereditary inclusion body myopathy (HIBM) is a young-adult onset autosomal recessive disorder caused by a hypomorphic rate limiting enzyme of sialic acid biosynthesis. The enzyme is UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, and is encoded by the GNE gene. HIBM causes slowly progressive muscle weakness and atrophy. Patients are typically diagnosed at 20-30 years of age, and most patients are incapacitated and wheelchair-confined by 30-50 years of age. Some sialic acid containing glycoproteins, including neural cell adhesion molecule (NCAM), are hyposialylated in HIBM muscle biopsy samples. We developed a method to allow detection of serum NCAM sialylation using Western blot, and tested serum samples from several patients and a HIBM mouse model. Preliminary results showed a clear difference in polysialylated and hyposialylated forms of NCAM extracted from serum, and showed NCAM is hyposialylated in HIBM serum samples. This initial finding may prove useful in reducing the need for serial muscle biopsies in HIBM treatment trials. Additional studies are underway to further validate this finding and to evaluate the specificity, reliability, and robustness of this potential serum biomarker for HIBM.

Loss of UDP‐ N ‐acetylglucosamine 2‐epimerase/ N ‐acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells

Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.

A preclinical trial of sialic acid metabolites on distal myopathy with rimmed vacuoles/ hereditary inclusion body myopathy, a sugar-deficient myopathy: a review

Distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy (hIBM), is a moderately progressive hereditary muscle disorder affecting young adults. DMRV/hIBM is characterized clinically by muscle atrophy and weakness initially involving the distal muscles, and pathologically by the presence of small angular fibers, formation of rimmed vacuoles and deposition of various proteins in the muscle fibers. This disease is known to be caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, which encodes the essential enzyme in sialic acid biosynthesis, leading to a reduction of sialic acid levels in the serum and skeletal muscles of affected patients. As it is a metabolic disease, metabolite supplementation is theoretically one of the therapeutic options. In this review, recent animal models for DMRV/hIBM are briefly characterized followed by a focus on the administration of sialic acid metabolites as a reliable therapeutic option to DMRV/hIBM with the following points highlighted: the property of compounds, the pharmacokinetic metabolism in vivo, and the therapeutic effects on the DMRV/hIBM mouse model.

Lessons from GNE-deficient embryonic stem cells: sialic acid biosynthesis is involved in proliferation and gene expression

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Inactivation of GNE causes early embryonic lethality. In this study, we analyzed wild-type and GNE-deficient embryonic stem cells from mice. We found for the first time that proliferation is directly correlated with GNE-expression and the cellular sialic acid concentration. Furthermore, we identified growth-related genes that are differentially expressed in GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells.

T.O.1 Sialic acid metabolites preclude the development of myopathic phenotype in the DMRV/hIBM mouse model

Distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (hIBM) is a moderately progressive autosomal recessive myopathy secondary to mutations in the GNE gene that encodes essential enzyme in sialic acid biosynthesis. It is characterized clinically by skeletal muscle atrophy and weakness initially affecting distal muscles. Muscle biopsy features include rimmed vacuoles, scattered angular fibers, and intracellular accumulation of amyloid and other proteins. To date, no therapy is available for this debilitating myopathy, mainly because precise understanding of the disease pathomechanism has been elusive.

Regulation and pathophysiological implications of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) as the key enzyme of sialic acid biosynthesis

The key enzyme for the biosynthesis of N-acetylneuraminic acid, from which all other sialic acids are formed, is the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). GNE is a highly conserved protein found throughout the animal kingdom. Its highest expression is seen in the liver and placenta. GNE is regulated by a variety of biochemical means, including tetramerization promoted by the substrate UDP-GlcNAc, phosphorylation by protein kinase C and feedback inhibition by CMP-Neu5Ac, which is defect in the human disease sialuria. GNE knock-out in mice leads to embryonic lethality, emphasizing the crucial role of this key enzyme for sialic acid biosynthesis. The metabolic capacity to synthesize sialic acid and CMP-sialic acid upon ManNAc loads is amazingly high. An additional characteristic of GNE is its interaction with proteins involved in the regulation of development, which might play a crucial role in the hereditary inclusion body myopathy. Due to the importance of increased concentrations of tumor-surface sialic acid, first attempts to find inhibitors of GNE have been successful.

The key enzyme of sialic acid biosynthesis (GNE) promotes neurite outgrowth of PC12 cells

Axonal outgrowth is a prerequisite for the development of the most complex organ, the brain. It depends partially on the attachment of sialic acid on glycans of (sialo)-glycoproteins expressed on the plasma membrane. In our study, we showed that nerve growth factor-induced neurite outgrowth of PC12-cells enhances the expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), the key enzyme for the biosynthesis of sialic acid. Furthermore, we could show that overexpression of GNE induces neurite outgrowth in PC12 cells. The neurite-outgrowth promoting activity of overexpressed GNE, however, does not lead to an increased biosynthesis of sialic acid. These data suggest a novel role of GNE during neurite outgrowth, which is independent to its specific enzymatic activity.

Allele‐specific silencing of the dominant disease allele in sialuria by RNA interference

Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.

G.P.13.09 Proteomic analysis of distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy (hIBM)

DMRV or hIBM is an autosomal recessive myopathy with an onset during young adulthood, characterized by the pathologic finding of rimmed vacuoles in skeletal muscles, the accumulation of various proteins, and the presence of tubulofilamentous inclusions. DMRV/hIBM is secondary to mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene which encodes for the key enzyme in sialic acid biosynthesis. We have shown that sialic acid is reduced in skeletal muscles obtained from DMRV/hIBM patients. Recently, we also developed a mouse model (Gne-/-hGNEV572L-Tg) for this disease, and have shown that muscle and other organs are hyposialylated. Up to this time, the role of hyposialylation in the pathomechanism of this myopathy has not been clarified. Further, proteins which are specifically involved in this myopathy have not been identified. We ventured on this study to clarify the proteomic characteristics of skeletal muscles from DMRV/hIBM patients compared to normal control. In order to investigate changes in expression of skeletal muscle proteins, proteomic analysis was done on skeletal muscles obtained from three patients with DMRV/hIBM and three healthy individuals, matched by age, gender, and site of biopsy. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Protein profiling was done by MALDI-TOF mass spectrometric analysis. In all three patients, several spots were differentially expressed. Upregulated proteins include human serum albumin and heat shock protein 27, among others. Interestingly, some spots were shifted towards the alkaline isoelectric point, which can infer a hyposialylated state; one among which was transferrin, a 76 kDa glycoprotein. Our results confirm that certain skeletal muscle proteins are differentially expressed and hyposialylated in DMRV/hIBM, and give clues to the pathomechanism of DMRV/hIBM. The precise role of these abnormal protein expressions in the development of symptoms in DMRV/hIBM remains to be elucidated.

Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N-acetylmannosamine

Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

Reduced sialylation status in UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE)-deficient mice

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Previously, we have shown that inactivation of the GNE by gene targeting causes early embryonic lethality in mice, whereas heterozygous GNE-deficient mice are vital. In this study we compared the amount of membrane-bound sialic acids of wildtype mice with those of heterozygous GNE-deficient mice. For that we quantified membrane-bound sialic acid concentration in various organs of wildtype- and heterozygous GNE-deficient mice. We found an organ-specific reduction of membrane-bound sialic acids in heterozygous GNE-deficient mice. The overall reduction was 25%. Additionally, we analyzed transferrin and polysialylated neural cell adhesion molecule (NCAM) by one- or two-dimensional gel electrophoresis. Transferrin-expression was unchanged in heterozygous GNE-deficient mice; however the isoelectric point of transferrin was shifted towards basic pH, indicating a reduced sialylation. Furthermore, the expression of polysialic acids on NCAM was reduced in GNE-deficient mice.

The collapsin response mediator protein 1 (CRMP-1) and the promyelocytic leukemia zinc finger protein (PLZF) bind to UDP- N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis

Sialic acids (Sia) are expressed as terminal sugars in many glycoconjugates. They are involved in a variety of cell–cell interactions and therefore play an important role during development and regeneration. UDP- N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE) is the key enzyme in the de novo synthesis of Sia and it is a regulator of cell surface sialylation. Inactivation of GNE in mice results in early embryonic lethality. Mutations in the GNE gene are of clinical relevance in hereditary inclusion body myopathy, but these mutations do not necessarily decrease the enzymatic activity of GNE. In this study, we searched for novel function of the GNE protein beside its enzymatic function in the Sia biosynthesis. We here report the identification of novel GNE-interacting proteins. Using a human prey matrix we identified four proteins interacting with GNE in a yeast two-hybrid assay. For two of them, the collapsin response mediator protein 1 and the promyelocytic leukemia zinc finger protein, we could verify protein–protein interaction with GNE.

Influence of UDP-GlcNAc 2-Epimerase/ManNAc Kinase Mutant Proteins on Hereditary Inclusion Body Myopathy

Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported to cause the disease. Ten of them, representing mutations in both functional domains of GNE, were recombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reduction in the two known GNE activities, thus revealing that mutations may also influence the function of the domain not harboring them. The extent of reduction strongly differs among the point mutants, ranging from only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activity in D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined by structural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis, respectively. We therefore generated models of the three-dimensional structures of the epimerase and the kinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-epimerase and glucokinase, respectively, and determined the localization of the HIBM mutations within these proteins. Whereas in the kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerase domain are mostly located at the protein surface. Otherwise, the different mutations result in different enzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role of the enzymatic function of GNE in the disease mechanism.