The story of the Sda antigen and of its cognate enzyme B4GALNT2: What is new?

The structure Siaα2,3(GalNAcß1,4)Gal- is the epitope of the Sda antigen, which is expressed on the erythrocytes and secretions of the vast majority of Caucasians, carried by N- and O-linked chains of glycoproteins, as well as by glycolipids. Sda is very similar, but not identical, to ganglioside GM2 [Siaα2,3(GalNAcß1,4)Galß1,4Glc-Cer]. The Sda synthase ß1,4 N-acetylgalactosaminyl transferase 2 (B4GALNT2) exists in a short and a long form, diverging in the aminoterminal domain. The latter has a very long cytoplasmic tail and displays a Golgi- as well as a post-Golgi localization. The biosynthesis of Sda is mutually exclusive with that of the cancer-associated sialyl Lewis antigens, whose structure is Siaα2,3Galß1,3/4(Fucα1,4/3)GlcNAc-. B4GALNT2 is down-regulated in colon cancer but patients with higher expression survive longer. In experimental systems, B4GALNT2 inhibits colon cancer progression,not only through inhibition of sialyl Lewis antigen biosynthesis. By contrast, in breast cancer B4GALNT2 is associated with malignancy. In colon cancer, the B4GALNT2 gene is regulated by multiple mechanisms, which include miRNA and transcription factor expression, as well as CpG methylation. In addition, Sda/B4GALNT2 regulates the susceptibility to infectious agents, the protection from muscle dystrophy, the activity of immune system in pregnancy and the immune rejection in xenotransplantation.

GALNT2, an O-glycosylating enzyme, is a critical regulator of radioresistance of non-small cell lung cancer: evidence from an integrated multi-omics analysis.

Radioresistance is the primary reason for radiotherapy failure in non-small cell lung cancer (NSCLC) patients. Glycosylation-related alterations are critically involved in tumor radioresistance. However, the relationship between glycosylation and NSCLC radioresistance is unclear. Here, we generated radioresistant NSCLC cell models by using fractionated irradiation. The aberrant glycosylation involved in NSCLC-related radioresistance was elucidated by transcriptomic, proteomic, and glycomic analyses. We conducted in vitro and in vivo investigations for determining the biological functions of glycosylation. Additionally, its downstream pathways and upstream regulators were inferred and verified. We demonstrated that mucin-type O-glycosylation and the O-glycosylating enzyme GALNT2 were highly expressed in radioresistant NSCLC cells. GALNT2 was found to be elevated in NSCLC tissues; this elevated level showed a remarkable association with response to radiotherapy treatment as well as overall survival. Functional experiments showed that GALNT2 knockdown improved NSCLC radiosensitivity via inducing apoptosis. By using a lectin pull-down system, we revealed that mucin-type O-glycans on IGF1R were modified by GALNT2 and that IGF1R could affect the expression of apoptosis-related genes. Moreover, GALNT2 knockdown-mediated in vitro radiosensitization was enhanced by IGF1R inhibition. According to a miRNA array analysis and a luciferase reporter assay, miR-30a-5p negatively modulated GALNT2. In summary, our findings established GALNT2 as a key contributor to the radioresistance of NSCLC. Therefore, targeting GALNT2 may be a promising therapeutic strategy for NSCLC.

Impacts of some clinicopathodemography and colorectal tissues key cell cycle and mucin stabilizing molecules on the metastasis trend in colorectal cancer patients.

BACKGROUND: We aimed to evaluate the various clinicopathodemographical, epidemiological, and molecular contributors to cumulatively worldwide metastatic colorectal cancer (CRC) in CRC patients from a highly populated area in northeastern Iran to pinpoint metastasis risk. METHODS: A retrospective clinical material-based cohort including a total of 6260 registered CRC patients, of whom 3829 underwent surgery, from regional university hospitals, during 2006-2016, were analyzed for the clinicopathodemographical aspects of age, sex, stage of CRC, history of smoking, type 2 diabetes (T2D), hypertension, body mass index (BMI), familial/occupational status, post-surgery survival period and mRNA/protein expression of mucin stabilizer (B3GALNT2), mucin I (MUC1), key cell cycle molecules (i.e., P53 and Ki67), and MMR-related genes. Factors were set to estimate the risk of metastatic CRC and mortality. RESULTS: Predominant adenocarcinomatous CRCs were found in colon. Post-surgery survival period of metastatic CRC patients was remarkably longer in patients aged > 50 compared to those aged < 50 years, and worse in females than males. B3GALNT2high, MUChigh, P53low, and Ki67high mRNA/protein expression in the metastatic stage III CRC along with T2D and hypertension were associated with increased metastasis/mortality, with more worsening in males, older, BMI > 25, urban residing, and employed individuals, indicative of non-genetic attributable factors. CONCLUSION: B3GALNT2, MUC1, and "Ki67" can be used as promising biomarkers for prognosis and early diagnosis of increasingly/predominantly non-genetic/environmental originated metastatic CRCs.

N-terminome analyses underscore the prevalence of SPPL3-mediated intramembrane proteolysis among Golgi-resident enzymes and its role in Golgi enzyme secretion.

Golgi membrane proteins such as glycosyltransferases and other glycan-modifying enzymes are key to glycosylation of proteins and lipids. Secretion of soluble Golgi enzymes that are released from their membrane anchor by endoprotease activity is a wide-spread yet largely unexplored phenomenon. The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by SPPL3 and released into cell culture supernatants. With high confidence, our study identifies more than 20 substrates of SPPL3, including entirely novel substrates. Notably, our N-terminome analyses provide a comprehensive list of SPPL3 cleavage sites demonstrating that SPPL3-mediated shedding of Golgi enzymes occurs through intramembrane proteolysis. Through the use of chimeric glycosyltransferase constructs we show that transmembrane domains can determine cleavage by SPPL3. Using our cleavage site data, we surveyed public proteome data and found that SPPL3 cleavage products are present in human blood. We also generated HEK293 knock-in cells expressing the active site mutant D271A from the endogenous SPPL3 locus. Immunoblot analyses revealed that secretion of select novel substrates such as the key mucin-type O-glycosylation enzyme GALNT2 is dependent on endogenous SPPL3 protease activity. In sum, our study expands the spectrum of known physiological substrates of SPPL3 corroborating its significant role in Golgi enzyme turnover and secretion as well as in the regulation of global glycosylation pathways.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Prenatal diagnosis of Walker-Warburg syndrome due to compound mutations in the B3GALNT2 gene.

BACKGROUND: Congenital hydrocephalus is one of the symptoms of Walker-Warburg syndrome that is attributed to the disruptions of the genes, among which the B3GALNT2 gene is rarely reported. A diagnosis of the Walker-Warburg syndrome depends on the clinical manifestations and the whole-exome sequencing after birth, which is unfavorable for an early diagnosis. METHODS: Walker-Warburg Syndrome was suspected in two families with severe fetal congenital hydrocephalus. Whole-exome sequencing and Sanger sequencing were performed on the affected fetuses. RESULTS: The compound heterozygous variants c.1A>G p.(Met1Val) and c.1151+1G>A, and c.1068dupT p.(D357*) and c.1052 T>A p.(L351*) in the B3GALNT2 gene were identified, which were predicted to be pathogenic and likely pathogenic, respectively. Walker-Warburg syndrome was prenatally diagnosed on the basis of fetal imaging and whole-exome sequencing. CONCLUSIONS: Our findings expand the spectrum of pathogenic mutations in Walker-Warburg syndrome and provide new insights into the prenatal diagnosis of the disease.

The O-glycosylating enzyme GALNT2 acts as an oncogenic driver in non-small cell lung cancer.

BACKGROUND: N-Acetylgalactosaminyltransferases (GALNTs), the enzymes that initiate mucin-type O-glycosylation, are closely associated with tumor occurrence and progression. However, a comprehensive analysis of GALNTs in non-small cell lung cancer (NSCLC) is lacking. METHODS: The expression profiles and prognostic values of the GALNT family members in NSCLC were analyzed using publicly available databases. Gain- and loss-of-function experiments were applied to assess the biological function of GALNT2 in NSCLC. High-throughput sequencing and bioinformatics approaches were employed to uncover the regulatory mechanism of GALNT2. RESULTS: Among the family members of GALNTs, only GALNT2 was frequently overexpressed in NSCLC tissues and was positively correlated with poor prognosis. In vitro assays showed that GALNT2 knockdown repressed NSCLC cell proliferation, migration, and invasion, but induced apoptosis and cell cycle arrest. Correspondently, GALNT2 overexpression exerted the opposite effects. In vivo experiments demonstrated that knockdown of GALNT2 restrained tumor formation in nude mice. Mechanistic investigations revealed that GALNT2 modified the O-glycosylation of ITGA5 and affected the activation of the PI3K/Akt and MAPK/ERK pathways. Further studies showed that miR-30d was a negative regulator of GALNT2. CONCLUSIONS: These findings suggest that GALNT2 is an oncogene in NSCLC and has the potential as a target for NSCLC therapy.

B4GALNT2 Controls Sda and SLex Antigen Biosynthesis in Healthy and Cancer Human Colon.

The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

Novel congenital disorder of O-linked glycosylation caused by GALNT2 loss of function.

Congenital disorders of glycosylation are a growing group of rare genetic disorders caused by deficient protein and lipid glycosylation. Here, we report the clinical, biochemical, and molecular features of seven patients from four families with GALNT2-congenital disorder of glycosylation (GALNT2-CDG), an O-linked glycosylation disorder. GALNT2 encodes the Golgi-localized polypeptide N-acetyl-d-galactosamine-transferase 2 isoenzyme. GALNT2 is widely expressed in most cell types and directs initiation of mucin-type protein O-glycosylation. All patients showed loss of O-glycosylation of apolipoprotein C-III, a non-redundant substrate for GALNT2. Patients with GALNT2-CDG generally exhibit a syndrome characterized by global developmental delay, intellectual disability with language deficit, autistic features, behavioural abnormalities, epilepsy, chronic insomnia, white matter changes on brain MRI, dysmorphic features, decreased stature, and decreased high density lipoprotein cholesterol levels. Rodent (mouse and rat) models of GALNT2-CDG recapitulated much of the human phenotype, including poor growth and neurodevelopmental abnormalities. In behavioural studies, GALNT2-CDG mice demonstrated cerebellar motor deficits, decreased sociability, and impaired sensory integration and processing. The multisystem nature of phenotypes in patients and rodent models of GALNT2-CDG suggest that there are multiple non-redundant protein substrates of GALNT2 in various tissues, including brain, which are critical to normal growth and development.

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2.

The type-I LacdiNAc (LDN; GalNAcß1-3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcß1-4GlcNAc) in N- and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a ß1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by Wisteria floribunda agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the B3GALNT2 gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their N-glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in vivo in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on N-glycans and suggest that type-I and type-II LDNs have different roles in vivo.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Mucin O-glycosylating enzyme GALNT2 facilitates the malignant character of glioma by activating the EGFR/PI3K/Akt/mTOR axis.

N-Acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that regulates the initial step of mucin O-glycosylation, has been reported to play a role in influencing the malignancy of various cancers. However, the mechanism through which it influences gliomas is still unknown. In the current study, the Cox proportional hazards model was used to select genes. Data obtained from The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC) of clinical specimens showed that increased GALNT2 expression levels were associated with an unfavorable prognosis and a higher tumor grade in human gliomas. Then, GALNT2 knockdown and overexpression were performed in glioma cell lines and verified by quantitative real-time PCR (qRT-PCR) and Western blotting. Functional assays demonstrated that GALNT2 was closely related to glioma cell proliferation, cycle transition, migration and invasion. Western blot analysis and lectin pull-down assays indicated that GALNT2 knockdown decreased the level of phosphorylated epidermal growth factor receptor (EGFR) and the expression of the Tn antigen on EGFR and affected the expression levels of p21, cyclin-dependent kinase 4 (CDK4), cyclinD1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) through the EGFR/PI3K/Akt/mTOR pathway. GALNT2 overexpression had the opposite effects. In vivo, the growth of orthotopic glioma xenografts in nude mice was distinctly inhibited by the expression of GALNT2 shRNA, and the tumors with GALNT2 shRNA exhibited less aggressiveness and reduced expression of Ki67 and MMP2. Overall, GALNT2 facilitates the malignant characteristics of glioma by influencing the O-glycosylation and phosphorylation of EGFR and the subsequent downstream PI3K/Akt/mTOR axis. Therefore, GALNT2 may serve as a novel biomarker and a potential target for future therapy of glioma.

The desirable donor pig to eliminate all xenoreactive antigens.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

Effect of lifelong carnitine supplementation on plasma and tissue carnitine status, hepatic lipid metabolism and stress signalling pathways and skeletal muscle transcriptome in mice at advanced age.

While strong evidence from clinical studies suggests beneficial effects of carnitine supplementation on metabolic health, serious safety concerns associated with carnitine supplementation have been raised from studies in mice. Considering that the carnitine doses in these mice studies were up to 100 times higher than those used in clinical studies, the present study aimed to address possible safety concerns associated with long-term supplementation of a carnitine dose used in clinical trials. Two groups of NMRI mice were fed either a control or a carnitine-supplemented diet (1 g/kg diet) from weaning to 19 months of age, and parameters of hepatic lipid metabolism and stress signalling and skeletal muscle gene expression were analysed in the mice at 19 months of age. Concentrations of free carnitine and acetylcarnitine in plasma and tissues were higher in the carnitine than in the control group (P<0·05). Plasma concentrations of free carnitine and acetylcarnitine were higher in mice at adult age (10 and 15 months) than at advanced age (19 months) (P<0·05). Hepatic mRNA and protein levels of genes involved in lipid metabolism and stress signalling and hepatic and plasma lipid concentrations did not differ between the carnitine and the control group. Skeletal muscle transcriptome analysis in 19-month-old mice revealed only a moderate regulation between carnitine and control group. Lifelong carnitine supplementation prevents an age-dependent impairment of plasma carnitine status, but safety concerns associated with long-term supplementation of carnitine at doses used in clinical trials can be considered as unfounded.

The high prolificacy of D'man sheep is associated with the segregation of the FecLL mutation in the B4GALNT2 gene.

Mutations in the FecL locus are associated with large variation in ovulation rate and litter size in the French Lacaune sheep breed. It has been shown that the B4GALNT2 gene within the FecL locus is most likely responsible for the high fecundity in the French breed. In this study, we have highlighted the segregation of the FecLL mutation within the B4GALNT2 gene in North African sheep breeds and notably in the highly prolific D'man breed. Genotyping of a sample of 183 Tunisian D'man individuals revealed a high frequency (0.65) of the prolific allele FecLL which was attributed to the adoption of a decades-old breeding strategy based on the selection of ewe lambs born from large litter size. Homozygous LL ewes showed a significantly increased litter size compared to heterozygous and non-carrier ewes (FecLL /FecLL  = 2.47 ± 0.09 vs. FecLL /FecL+  = 2.23 ± 0.09, p < 0.05 and FecL+ /FecL+  = 1.93 ± 0.18, p < 0.01). The presence of the FecLL polymorphism in both D'man and Lacaune breeds argues for an ancestral origin of this mutation and brings an answer to the old question of the genetic determinism of the extreme prolificacy of the D'man ewes. The results of this study can help to establish planned genotype-based mating allowing both higher profit for the breeders and an optimal management of the FecLL mutation in D'man sheep populations.

Comparative Transcriptomics Identifies Novel Genes and Pathways Involved in Post-Traumatic Osteoarthritis Development and Progression.

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.

Evaluating the maintenance of disease-associated variation at the blood group-related gene B4galnt2 in house mice.

BACKGROUND: B4galnt2 is a blood group-related glycosyltransferase that displays cis-regulatory variation for its tissue-specific expression patterns in house mice. The wild type allele, found e.g. in the C57BL/6 J strain, directs intestinal expression of B4galnt2, which is the pattern observed among vertebrates, including humans. An alternative allele class found in the RIIIS/J strain and other mice instead drives expression in blood vessels, which leads to a phenotype similar to type 1 von Willebrand disease (VWD), a common human bleeding disorder. We previously showed that alternative B4galnt2 alleles are subject to long-term balancing selection in mice and that variation in B4galnt2 expression influences host-microbe interactions in the intestine. This suggests that the costs of prolonged bleeding in RIIIS/J allele-bearing mice might be outweighed by benefits associated with resistance against gastrointestinal pathogens. However, the conditions under which such trade-offs could lead to the long-term maintenance of disease-associated variation at B4galnt2 are unclear. RESULTS: To explore the persistence of B4galnt2 alleles in wild populations of house mice, we combined B4galnt2 haplotype frequency data together with a mathematical model based on an evolutionary game framework with a modified Wright-Fisher process. In particular, given the potential for a heterozygote advantage as a possible explanation for balancing selection, we focused on heterozygous mice, which express B4galnt2 in both blood vessels and the gastrointestinal tract. We show that B4galnt2 displays an interesting spatial allelic distribution in Western Europe, likely due to the recent action of natural selection. Moreover, we found that the genotype frequencies observed in nature can be produced by pathogen-driven selection when both heterozygotes and RIIIS/J homozygotes are protected against infection and the fitness cost of bleeding is roughly half that of infection. CONCLUSION: By comparing the results of our models to the patterns of polymorphism at B4galnt2 in natural populations, we are able to recognize the long-term maintenance of the RIIIS/J allele through host-pathogen interactions as a viable hypothesis. Further, our models identify that a putative dominant-, yet unknown protective function of the RIIIS/J allele appears to be more likely than a protective loss of intestinal B4galnt2 expression in RIIIS/J homozygotes.

Genotyping of friesian horses to detect a hydrocephalus-associated c.1423C>T mutation in B3GALNT2 using PCR-RFLP and PCR-PIRA methods: Frequency in stallion horses in México.

Hydrocephalus in Friesian horses is an autosomal recessive hereditary disease that can result in an abortion, a stillbirth, or euthanization of a newborn foal. Here, the hydrocephalus-associated c.1423C > T mutation in B3GALNT2 gene was detected with PCR-RFLP and PCR-PIRA methods for horse genotyping. A preliminary genotyping survey was performed on 83 randomly selected Friesian stallion horses to determine the current allele frequency in Mexico. The frequency of the mutant T allele was 9.6%.

GALNT2 effect on HDL-cholesterol and triglycerides levels in humans: Evidence of pleiotropy?

A wide range of studies both in humans and animal models point GALNT2 as a shaper of serum HDL-C and TG levels. Available data in humans indicate that, while under conditions of extreme GALNT2 loss-of-function HDL-C is the main target, a fine-tuning of GALNT2 changes is mostly associated with TG levels. Understanding whether different degrees of GALNT2 change do modulate different serum lipid fractions and, if so, addressing the mechanisms underlying such pleiotropic effects has the potential not only to improve our understanding of HDL-C and TG metabolism, but also to make GALNT2 becoming a target for treating atherogenic dyslipidemia and related clinical events.