In the current study we explored GNE isoform expression in human tissues and compared structural differences between hGNE1 and six other human GNE isoforms by molecular modeling. In addition to the previously described 3 human GNE isoforms, database and PCR analysis yielded an additional 5 hGNE isoforms, summarized in . Since the commercially available GNE antibodies do not work well on Western blotting, we were unable to confirm expression of each transcript at the protein level. In our predictions below, we assumed each isoform transcript produces a protein.
Tissue analysis showed differential expression of the 8 hGNE isoform encoding transcripts. hGNE1 appeared to be the major isoform, with mRNA expression in all tissues tested. hGNE6 was the only other isoform whose mRNA was expressed in all tested tissues (although at low levels). hGNE6 likely only has kinase activity, since it lacks part of the epimerase functional domain (skipping of in-frame exon 5 compared to hGNE1). We identified 4 other hGNE transcripts (hGNE5, hGNE7, hGNE8) that lacked exon 5 and, therefore, are predicted to display only kinase activity. Expression of hGNE5 was very low with conventional PCR (), and no hGNE5-specific Taqman assay could be designed, limiting our further pursuit of hGNE5 expression. hGNE7 and hGNE8 followed a similar tissue-distribution pattern and were expressed at low levels in selected human tissues (, ). Interestingly, expression of hGNE2 and hGNE3 coding transcripts followed the same tissue-distribution pattern as hGNE7 and hGNE8, indicating that they may be regulated together. We did not detect hGNE4 coding mRNA transcripts in tested tissues, and further analysis showed that this variant was submitted as a mutation identified in a DMRV patient (GenBank EU093084), instead of a novel isoform. We list this variant because a GenBank accession number is ascribed to this isoform, listed in GenBank as variant 5, but we did not further analyze hGNE4 by molecular modeling.
Our cDNA analysis of a variety of mouse tissues identified transcripts encoding the two previously described mouse isoforms mGne1 and mGne2, which have high homologies of 98.5% and 96.8% to hGNE1 and hGNE2, respectively (21
). Orthologs of the other human isoforms do not appear to exist in mouse tissues, perhaps due to an evolutionary mechanism.
The highest hGNE
mRNA expression levels occurred in the liver and placenta. Liver is the major organ of sialic synthesis and was shown to display high GNE enzymatic activity (31
). High hGNE expression in placenta may be related to an essential role of sialic acid during development (31
). The lowest hGNE mRNA expression was found in skeletal muscle, as previously described (31
), which may explain why skeletal muscle is the only affected tissue in HIBM\DMRV patients. A slight decrease in GNE enzymatic activities in HIBM\DMRV muscle may have a larger effect on sialic acid supply in muscle than in other tissues that have higher natural GNE expression and enzyme activity levels. The effect of HIBM\DMRV patient mutations on hGNE isoform expression remains to be determined.
GNE epimerase and kinase enzymatic activities were reported for the hGNE-1,-2, and -3 isoforms (22
). Recombinantly expressed hGNE1 contained the highest epimerase activity (~1100 mU/mg) and existed in the tetrameric (full enzyme activity) and dimeric (kinase activity only) states. hGNE2 displayed an 80% reduction in epimerase activity compared to hGNE1 (~200 mU/mg) explained by its existence predominantly in the dimeric state. hGNE3 had no epimerase activity, likely because a part of the epimerase domain encoded by exon 3 is not expressed in this isoform. The oligomeric state of hGNE3 could not be determined due to low expression levels. hGNE-1,-2, and -3 all displayed high kinase activities (ranging from 2500–3500 mU/mg). Enzyme activities for the isoforms hGNE-5,-6,-7, and -8 are not known, but it is likely that they have no epimerase activity, since they lack exon 5, encoding part of the epimerase domain. In fact, our modeling studies of these isoforms, also predict diminished or absent epimerase activity. hGNE-5,-6,-7, and -8 likely have residual kinase activity, since they express the kinase domain encoding C-terminal exons. These results suggest that hGNE1 (with high epimerase activity) has a major role in sialic acid production. The other human isoforms (with primarily kinase activity) may assist in the crucial regulation of cellular sialic acid levels (22
For molecular modeling all considered human isoforms were analyzed in 3 groups. First, the 31-residue N-terminal extension compared to the hGNE1 isoform, occurring in hGNE2 and hGNE7 was analyzed. This extension produces α-helices which extend the Rossmann fold. Since similar extended Rossman folds N-terminal of the nucleotide binding domain are found in S. achanthias
muscle lactate dehydrogenase, P. aeruginosa
UDP-N-acetylglucosamine 4-epimerase and other proteins, we assume the additional N-terminal fragments of hGNE2 and hGNE7 do not hinder the active sites of the GNE enzyme and will likely not eliminate epimerase activity. In fact, this was confirmed by enzyme activity measurements in recombinantly expressed hGNE2, which displayed 80% epimerase activity and similar ManNAc kinase activity of that of GNE1 (22
). The 31-residue extension at N-termini of hGNE2 and hGNE7 showed amino acid similarities with ATP-binding fragments of some kinases and helicases (). Based on homology to SGK1 and other kinases, the cysteine C12 residue in the 31-residue extension may be a candidate for disulfide bond formation. It was previously suggested that the 31-residue extension in GNE2 may have a regulatory role in fine-tuning sialic acid synthesis (21
Second, we considered the 55 residue N-terminal deletion, and a novel 50-residue extension in the hGNE3 and hGNE8 isoforms. Interestingly, the size and secondary structure (alternating α-helices and β-strands; ) of the deleted and extension fragments are similar, indicating that the extension may serve as a replacement for the deletion. In addition, the novel 50-residue extension has similarity to other proteins containing the Rossmann fold such as deaminases, glycosyltransferases, α-1,2-mannosidase, ATPase, and guardian (). These findings indicate that the N-terminal modification of hGNE3 and hGNE8 may not affect the overall fold of the protein, compared to hGNE1. However, epimerase function of the hGNE3 and hGNE8 isoforms is predicted to be diminished or absent, since the deleted 55 residue fragment contains several amino acids important for enzymatic function, including substrate binding. In homologous bacterial epimerase enzymes, this region is part of an active and allosteric site. In addition, it was shown that deletion of the N-terminal 39 amino acids of hGNE1 leads to complete loss of epimerase activity with 22% of the kinase activity retained (32
). Indeed, activity measurements of the recombinant hGNE3 enzyme confirmed loss of epimerase enzymatic activity while ManNAc kinase activity was retained, likely due to presence of the unaffected kinase domain (21
Lastly, in addition to a N-terminal modification, a 53-residue deletion occurring in hGNE5, hGNE6, hGNE7, and hGNE8 was considered. The deleted region contains a predicted catalytic residue and is assigned a role in the binding of UDP-GlcNAc and stabilization of the UDP intermediate (displayed in ). Deletion of this fragment likely results in abolished epimerase activity, while ManNAc kinase activity may remain present. No enzymatic studies have been performed for these isoforms. The hGNE5 isoform contains the above mentioned 53- residue deletion (encoded by exon 5) as well as a 110-residue N-terminal deletion (and no extension) compared to hGNE1. Therefore hGNE5 is also predicted to have abolished epimerase activity with residual kinase activity, which remains to be confirmed by enzymatic activity measurements.
We previously established that the major GNE1 isoform consists of 7-stranded parallel β-sheets are sandwiched between a total of 7 α-helices in the N-terminal domain and 6-stranded β-sheet surrounded by a total of 7 α-helices in the C-terminal domain (11
). The epimerase encoding region of GNE consists of two domains, domain I (N-terminal) and domain II (Cterminal) (). These two domains show structural similarities and both have a topology similar to the Rossman nucleotide binding fold. Interestingly, the two major deleted fragments in the hGNE isoforms, one 55-residue deletion at the N-terminus of the domain I in the hGNE3 and hGNE8 isoforms, and the other a 53-residue deletion at the N-terminus of the domain II in the hGNE5, hGNE6, hGNE7 and hGNE8 isoforms (, ), also show structural similarities between each other. Both deleted fragments consist of βαβα secondary structure elements at their N-termini, and both contain similarly located conserved active site residue(s) and non-conserved bacterial allosteric site amino acid residues. It is possible that evolutionary evolvement of the different hGNE isoforms illustrates a process of the elimination of the bacterial allosteric site from the enzyme, as well as further evolution of the mammalian bifunctional enzyme from two separate bacterial epimerase and kinase enzymes.
Evolution displays several mechanisms by which the number of cellular functions can be increased, without increasing the number of genes (38
). These include expression of multiple mRNAs from one gene (by alternative mRNA transcription or alternative splicing), multiple translation products from one gene (by translation initiation from alternative in-frame start codons), tissue-specific mRNA or protein expression, protein targeting to different cellular compartments, post-translational protein modification (through phosphorylation, glyscosylation, etc.), and oligomer formation, among others. It appears as if the GNE
gene is an excellent example of being subject to some of the above mechanisms. First, the GNE
gene displays an increased number of mRNA transcripts through evolution; while the mouse Gne
gene only has 2 mRNA transcripts, the human GNE
gene produces at least 7 different transcripts (formed by either alternative splicing or alternative transcription, or both). Second, GNE
mRNA transcripts are tissue-specific expressed, possibly due to tissue-specific demands for sialylation or other regulatory processes independent of sialic acid production, such as sialyltransferase expression, ganglioside production and modulation of proliferation and apoptosis (39
). Third, the different hGNE isoforms (if translated) may have different localization within the cell. hGNE1 can be localized to the nucleus, cytoplasm, or the Golgi-region, perhaps targeted by a putative leucine rich nuclear export signal, located within amino acids 121–140 of hGNE1 (40
). This region is highly conserved and is not deleted or modified in any of the other human isoforms. Fourth, hGNE has several potential phosphorylation sites that may affect function (41
). Some predicted phosphorylation sites (by NetPhos 2.0) are deleted (such as Thr34, Tyr22, Tyr54 of hGNE1 are deleted in hGNE3, hGNE5 and hGNE8) or added (such as Ser11 and Tyr4 of hGNE2 and hGNE7 are not present in hGNE1) in the different hGNE isoforms compared to hGNE1. And finally, GNE exists in two major oligomeric states, tetramers and dimers, which engage in dynamic interplay with monomers and higher aggregates. As a monomer, GNE has no enzymatic activity, its dimer exhibits only ManNAc kinase activity, and the tetrameric state displays both UDP-GlcNAc 2-epimerase and ManNAc kinase activities (42
). In addition, the fully functional tetrameric state of GNE is stabilized by ligands of the epimerase domain, UDP-GlcNAc and CMP-Neu5Ac. Our molecular modeling studies may contribute to predictions of isoform involvement in oligomer formation and enzyme activity of oligomeric complexes.
In summary, our human GNE isoform studies revealed that hGNE1 exist as ubiquitously expressed, major isoform, while the hGNE2-8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. Genetic analysis of the novel coding regions of hGNE exons 1–3 are warranted in patients with HIBM\DMRV, especially those in which only one GNE mutation could be identified. These expression and structural prediction data may contribute to elucidation of the complex functional and regulatory mechanisms of GNE, as well as to further elucidation of the pathology and treatment strategies of the human GNE-opathies sialuria and HIBM\DMRV, as well as of other disorders of hyposialylation.
shows primer sequences for PCR amplification of the human GNE
). Table S2
shows TaqMan primer and probe sequences for qPCR to determine expression of hGNE