CMP-N-acetylneuraminic Acid Hydroxylase Research Articles

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah-/-mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah-/-mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection

BackgroundThe use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins’ chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known.ResultsWe mutated mouse CMAH in the NOD/scid-IL2Rγc−/− (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8.ConclusionNSG-cmah−/− mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.

Phylogenetic Distribution of CMP-Neu5Ac Hydroxylase (CMAH), the Enzyme Synthetizing the Proinflammatory Human Xenoantigen Neu5Gc.

The enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid present on the cell surface proteins of most deuterostomes. The CMAH gene is thought to be present in most deuterostomes, but it has been inactivated in a number of lineages, including humans. The inability of humans to synthesize Neu5Gc has had several evolutionary and biomedical implications. Remarkably, Neu5Gc is a xenoantigen for humans, and consumption of Neu5Gc-containing foods, such as red meats, may promote inflammation, arthritis, and cancer. Likewise, xenotransplantation of organs producing Neu5Gc can result in inflammation and organ rejection. Therefore, knowing what animal species contain a functional CMAH gene, and are thus capable of endogenous Neu5Gc synthesis, has potentially far-reaching implications. In addition to humans, other lineages are known, or suspected, to have lost CMAH; however, to date reports of absent and pseudogenic CMAH genes are restricted to a handful of species. Here, we analyze all available genomic data for nondeuterostomes, and 322 deuterostome genomes, to ascertain the phylogenetic distribution of CMAH. Among nondeuterostomes, we found CMAH homologs in two green algae and a few prokaryotes. Within deuterostomes, putatively functional CMAH homologs are present in 184 of the studied genomes, and a total of 31 independent gene losses/pseudogenization events were inferred. Our work produces a list of animals inferred to be free from endogenous Neu5Gc based on the absence of CMAH homologs and are thus potential candidates for human consumption, xenotransplantation research, and model organisms for investigation of human diseases.

N-glycolylneuraminic acid on human epithelial cells prevents entry of influenza A viruses that possess N-glycolylneuraminic acid binding ability.

Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.

220 LENGTH OF DONOR DNA HOMOLOGY TO FACILITATE BI-ALLELIC GENE TARGETING DURING TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-MEDIATED GENE TARGETING

Gene editing techniques are increasing in their availability and ease of use. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the efficiency of targeting. We have previously produced CMP-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFN) in combination with donor DNA containing a total length of homology of 1600 bp (800-bp homology on each arm). A second KO was desired in the CMAH –/– genetic background, GGTA1. We used this opportunity to evaluate the effect of total donor homology on transcription activator-like effector nuclease (TALEN) mediated bi-allelic modification. Donor DNA was designed to symmetrically flank the predicted TALEN cleavage site in GGTA1 and was varied in length of total homology (40, 100, 200, 460, 890, and 1800 bp). The TALEN constructs and donor DNA were introduced into fibroblast cells by a lipid-based transfection method. Each donor DNA was co-transfected with a pair of TALEN constructs. In addition, the TALEN pair alone and the longest donor DNA alone were separately transfected to evaluate error-prone non-homologous end joining (NHEJ) repair or background HR, respectively. Five days post transfection, cells were evaluated for the ablation of the α-1,3 gal epitope, which results from bi-allelic disruption of GGTA1. Lectin IB-4 (FITC labeled) was used to detect the presence/absence of the epitope. If a bi-allelic modification to the GGTA1 gene was accomplished, specific binding of the lectin would not occur; therefore, a lack of FITC was evaluated. Donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (no donor DNA) to a maximum of 3% (1800 bp). Inclusion of homologous donor DNA in TALEN transfections facilitates HR that competes with error-free NHEJ. The use of donor DNA with TALEN therefore can provide for increased bi-allelic disruption and predictability in the sequence of the resulting mutation.

Enhanced T Cell Function in a Mouse Model of Human Glycosylation

Clinical evidence for a more active immune response in humans compared with our closest hominid relative, the chimpanzee, includes the progression of HIV infection to AIDS, hepatitis B- and C-related inflammation, autoimmunity, and unwanted harmful immune responses to viral gene transfer vectors. Humans have a unique mutation of the enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH), causing loss of expression of the sialic acid Neu5Gc. This mutation, occurring 2 million years ago, likely altered the expression and function of ITIM-bearing inhibitory receptors (Siglecs) that bind sialic acids. Previous work showed that human T cells proliferate faster than chimpanzee T cells upon equivalent stimulation. In this article, we report that Cmah(-/-) mouse T cells proliferate faster and have greater expression of activation markers than wild-type mouse T cells. Metabolically reintroducing Neu5Gc diminishes the proliferation and activation of both human and murine Cmah(-/-) T cells. Importantly, Cmah(-/-) mice mount greater T cell responses to an adenovirus encoding an adeno-associated virus capsid transgene. Upon lymphocytic choriomeningitis virus infection, Cmah(-/-) mice make more lymphocytic choriomeningitis virus-specific T cells than WT mice, and these T cells are more polyfunctional. Therefore, a uniquely human glycosylation mutation, modeled in mice, leads to a more proliferative and active T cell population. These findings in a human-like mouse model have implications for understanding the hyperimmune responses that characterize some human diseases.

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.

Alpha 1,3-Galactosyltransferase Deficiency in Pigs Increases Sialyltransferase Activities That Potentially Raise Non-Gal Xenoantigenicity

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

Human CMP-N-Acetylneuraminic Acid Hydroxylase Is a Novel Stem Cell Marker Linked to Stem Cell-Specific Mechanisms

Human stem cells contain substantial amounts of the xenoantigen N-glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP-N-acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell-specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co-occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of beta-catenin in both CMAH overexpressing cells and bone marrow-derived mesenchymal stem cells, thereby inactivating Wnt/beta-catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc-induced alteration of crucial stem cell-specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells.

CMP-N-Acetylneuraminic Acid Hydroxylase Is Exclusively Inactive in Humans

We cloned cDNAs for mouse and human CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylases and showed that the human CMP-NeuAc hydroxylase protein is inactive because of a partial deletion in the hydroxylase gene. We report here that no other active CMP-NeuAc hydroxylases are present in humans. Southern blot analysis showed that the human homologue of the mouse CMP-NeuAc hydroxylase is one gene in the human genome and no other homologues of the mouse hydroxylase exist in human genome. The mouse and the human CMP-NeuAc hydroxylases were mapped to chromosome 13A3 and chromosome 6p22, respectively, by fluorescencein situhybridization. The chromosomal location of the human hydroxylase is syntenic to that of the mouse hydroxylase. These results demonstrate that the human CMP-NeuAc hydroxylase is the only homologue of the mouse hydroxylase, and CMP-NeuAc hydroxylase is exclusively inactive in humans.

Reaction mechanism underlying CMP-N-acetylneuraminic acid hydroxylation in mouse liver: formation of a ternary complex of cytochrome b5, CMP-N-acetylneuraminic acid, and a hydroxylation enzyme.

We have proposed that CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is mediated by an electron transport system consisting of cytochrome b5 (b5), b5 reducing factor(s), and CMP-NeuAc hydroxylase, all of which have been detected in the cytosolic fraction of mouse liver [Kozutsumi, Y., Kawano, T., Yamakawa, T., & Suzuki, A. (1990) J. Biochem. 108, 704-706]. In order to elucidate the reaction mechanism underlying CMP-NeuAc hydroxylation, the interaction between b5 and the hydroxylase was studied using a b5-immobilized affinity column. The enzyme activity was retarded on the b5 column in the presence of the substrate, CMP-NeuAc, but not in the presence of the reaction product, CMP-N-glycolyneuraminic acid (CMP-NeuGc). These findings suggest that the binding of CMP-NeuAc to CMP-NeuAc hydroxylase changes the conformation of the enzyme so as to construct a recognition site for b5, followed by the formation of a ternary complex through this domain. Then the transport of electrons from NAD(P)H to the enzyme through b5 takes place, CMP-NeuAc is converted to CMP-NeuGc, and finally the ternary complex dissociates into its components to release CMP-NeuGc. It is known that a soluble form of b5 is abundant in erythrocytes and is synthesized from a mRNA different from that for the microsomal form of b5. In order to determine the origin of b5 detected in the cytosolic fraction of mouse liver, the molecular forms of b5 mRNA expressed in mouse liver were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)