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1.
Biochem Biophys Res Commun ; 703: 149610, 2024 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-38359610

RESUMEN

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.


Asunto(s)
Acetilglucosamina , Galactosiltransferasas , Sialiltransferasas , Animales , Humanos , Ratones , Acetilglucosamina/metabolismo , Drosophila/metabolismo , Galactosiltransferasas/genética , Glicosiltransferasas , Células HEK293 , Polisacáridos , Receptores Notch/metabolismo , Sialiltransferasas/genética
2.
Anim Biotechnol ; 33(6): 1056-1064, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33427026

RESUMEN

Beta 1,4-galactosyltransferase-I gene (B4GALT1) is an important candidate gene for milk performance traits, encodes catalytic part of lactose synthesis. Main objectives of present study is identification of single-nucleotide polymorphism in exon 1 and 2 region of B4GALT1 and to find significant association of genetic variants with milk performance traits in crossbred cattle of Kerala. The study was conducted on two hundred crossbred cattle maintained at various farms of Kerala Veterinary and Animal Sciences University, India. Genomic DNA was isolated and polymorphism of gene were detected by Single Strand Confirmation Polymorphism. Genotype and allelic frequency were estimated. Chi-square analysis revealed that screened population is under Hardy Weinberg equilibrium. Nucleotide sequence analysis revealed a novel non-synonymous single-nucleotide variation (T94A) in exon 1 and a non-synonymous mutation of T97C in exon 2 of B4GALT1 gene in the screened cattle population. Association analysis of genetic variants was done with milk production traits and major non-genetic factors using fixed models. Different genetic variants of B4GALT1 was significantly associated with 305 days milk yield, lactose, protein percent. Study indicates existence of genetic variability in B4GALT1 gene on crossbred cattle of Kerala and suggests a scope of considering genetic variants of B4GALT1in selection strategies.


Asunto(s)
Leche , Polimorfismo de Nucleótido Simple , Bovinos/genética , Animales , Femenino , Leche/metabolismo , Polimorfismo de Nucleótido Simple/genética , Lactosa/metabolismo , Frecuencia de los Genes/genética , Fenotipo , Genotipo , Lactancia/genética
3.
Int J Mol Sci ; 23(23)2022 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-36499368

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lung that involves the pulmonary interstitium. The disease may rapidly progress, leading to respiratory failure, and the long-term survival is poor. There are no accurate biomarkers available so far. Our aim was to evaluate the expression of the B4GALT1 in patients with IPF. Analysis of B4GALT1 gene expression was performed in silico on two gene sets, retrieved from the Gene Expression Omnibus database. Expression of B4GALT1 was then evaluated, both at the mRNA and protein levels, on lung specimens obtained from lung biopsies of 4 IPF patients, on one IPF-derived human primary cell and on 11 cases of IPF associated with cancer. In silico re-analysis demonstrated that the B4GALT1 gene was overexpressed in patients and human cell cultures with IPF (p = 0.03). Network analysis demonstrated that B4GALT1 upregulation was correlated with genes belonging to the EMT pathway (p = 0.01). The overexpression of B4GALT1 was observed, both at mRNA and protein levels, in lung biopsies of our four IPF patients and in the IPF-derived human primary cell, in other fibrotic non-lung tissues, and in IPF associated with cancer. In conclusion, our results indicate that B4GALT1 is overexpressed in IPF and could represent a novel marker of this disease.


Asunto(s)
Fibrosis Pulmonar Idiopática , Neoplasias , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/patología , Biomarcadores/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias/metabolismo
4.
J Cell Mol Med ; 24(20): 12032-12043, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32902124

RESUMEN

Multiple mechanisms contribute to the survival and growth of metastatic castration-resistant prostate cancer (mCRPC) cells without androgen, including androgen receptor splice variants (AR-V) and de novo intratumoral androgen synthesis. AKR1C3 is a critical androgenic enzyme that plays different roles in mCRPC, such as an EMT driver or AR coactivator. However, the relationship and regulatory mechanisms between AKR1C3 and AR-V remain largely unknown. In this study, we observed a positive correlation between AKR1C3 and AR-V7 staining in tissues from prostate rebiopsy at mCRPC. Mechanistically, AKR1C3 interacts with AR-V7 protein in CRPC cells, which can reciprocally inhibit AR-V7 and AKR1C3 protein degradation. Biologically, this complex is essential for in vitro and in vivo tumour growth of CRPC cells after androgen deprivation as it represses B4GALT1, a unique tumour suppressor gene in PCa. Together, this study reveals AKR1C3/AR-V7 complex as a potential therapeutic target in mCRPC.


Asunto(s)
Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Galactosiltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular , Galactosiltransferasas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Biológicos , Unión Proteica , Estabilidad Proteica , Transcripción Genética , Ubiquitina/metabolismo
5.
Metab Eng ; 61: 301-314, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32663509

RESUMEN

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Glicosiltransferasas , Ingeniería Metabólica , Polisacáridos , Animales , Células CHO , Cricetulus , Glicosilación , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/genética , Polisacáridos/biosíntesis , Polisacáridos/genética
6.
Glycoconj J ; 37(6): 767-775, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32926333

RESUMEN

ß1,4-GalT1 is a type II membrane glycosyltransferase. It catalyzes the production of lactose in the lactating mammary gland and is supposedly also involved in the galactosylation of terminal GlcNAc of complex-type N-glycans. In-vitro studies of the bovine ß4Gal-T1 homolog showed that replacing a single residue of tyrosine with leucine at position 289 alters the donor substrate specificity from UDP-Gal to UDP-N-acetyl-galactosamine (UDP-GalNAc). The effect of this peculiar change in ß1,4GalT1 specificity was investigated in-vivo, by generating biallelic Tyr286Leu ß1,4GalT1 mice using CRISPR/Cas9 and crossbreeding. Mice bearing this mutation showed no appreciable defects when compared to wild-type mice, with the exception of biallelic female B4GALT1 mutant mice, which were unable to produce milk. The detailed comparison of wild-type and mutant mice derived from liver, kidney, spleen, and intestinal tissues showed only small differences in their N-glycan pattern. Comparable N-glycosylation was also observed in HEK 293 wild-type and knock-out B4GALT1 cells. Remarkably and in contrast to the other analyzed tissue samples, sialylation and galactosylation of serum N-glycans of biallelic Tyr286Leu GalT1 mice almost disappeared completely. These results suggest that ß1,4GalT1 plays a special role in the synthesis of serum N-glycans. The herein described Tyr286Leu ß1,4GalT1 mutant mouse model may, therefore, prove useful in the investigation of the mechanism which regulates tissue-dependent galactosylation.


Asunto(s)
Galactosa/metabolismo , Galactosiltransferasas/genética , Polisacáridos/sangre , Animales , Bovinos , Femenino , Galactosiltransferasas/metabolismo , Glicosilación , Células HEK293 , Humanos , Lactancia/genética , Ratones , Polimorfismo de Nucleótido Simple/genética , Polisacáridos/genética , Especificidad por Sustrato
7.
J Inherit Metab Dis ; 43(3): 611-617, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31800099

RESUMEN

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying the effects of defective glycosylation on plasma lipids in patients with B4GALT1-CDG, caused by a mutation in B4GALT1 with defective N-linked glycosylation. We studied plasma lipids, cholesteryl ester transfer protein (CETP) glyco-isoforms with isoelectric focusing followed by a western blot and CETP activity in three known B4GALT1-CDG patients and compared them with 11 age- and gender-matched, healthy controls. B4GALT1-CDG patients have significantly lowered non-high density lipoprotein cholesterol (HDL-c) and total cholesterol to HDL-c ratio compared with controls and larger HDL particles. Plasma CETP was hypoglycosylated and less active in B4GALT1-CDG patients compared to matched controls. Our study provides insight into the role of protein glycosylation in human lipoprotein homeostasis. The hypogalactosylated, hypo-active CETP found in patients with B4GALT1-CDG indicates a role of protein galactosylation in regulating plasma HDL and LDL. Patients with B4GALT1-CDG have large HDL particles probably due to hypogalactosylated, hypo-active CETP.


Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Trastornos Congénitos de Glicosilación/genética , Galactosiltransferasas/genética , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Proteínas de Transferencia de Ésteres de Colesterol/genética , Trastornos Congénitos de Glicosilación/metabolismo , Femenino , Glicosilación , Homocigoto , Humanos , Lactante , Masculino , Mutación
8.
J Cell Physiol ; 234(10): 18524-18534, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30912138

RESUMEN

Here, an RNA-sequencing assay revealed long noncoding RNAs (lncRNAs) with an ectopic expression between colon cancer (CC) and normal colon epithelial cells, in which lncRNA B4GALT1-AS1 exhibited the highest change. A 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay indicated that B4GALT1-AS1 knockdown had no effect on CC cell viability, however, cell clone formation analysis showed that B4GALT1-AS1 knockdown attenuated the capacity of cell clone formation. Additionally, gene set enrichment analysis of this data set revealed that positive enrichment of stem cell-differentiated signatures and negative embryonic stem cell function and adult tissue stem module were observed in CC cells with B4GALT1-AS1 knockdown. Furthermore, B4GALT1-AS1 knockdown suppressed the stemness-marker expression, the ability of cell spheroid formation, and ALDH1 activity in CC cells. Mechanistically, RNA-sequencing data found that the Hippo pathway in cancer was shown on pathways mostly upregulated by B4GALT1-AS1 knockdown, and B4GALT1-AS1 directly bound to the yes-associated protein (YAP), a downstream executor of the Hippo pathway, and B4GALT1-AS1 knockdown promoted the nuclear cytoplasm translocation of YAP and decreased YAP transcriptional activity. Notably, YAP overexpression attenuated the inhibitory effects mediated by B4GALT1-AS1 knockdown. Our results identify the direct binding of lncRNA B4GALT1-AS1 to YAP, which is responsible for CC cell stemness.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Movimiento Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células Madre Neoplásicas/metabolismo , ARN Largo no Codificante/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Células Clonales , Transición Epitelial-Mesenquimal , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP
9.
BMC Vet Res ; 15(1): 457, 2019 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-31852463

RESUMEN

BACKGROUND: Milk sialylated oligosaccharides (SOS) play crucial roles in many biological processes. The most abundant free SOS in goat's milk are 3'sialyllactose (3'-SL), 6'sialyllactose (6'-SL) and disialyllactose (DSL). The production of these molecules is determined genetically by the expression of glycosyltransferases and by the availability of nucleotide sugar substrates, but the precise mechanisms regulating the differential patterns of milk oligosaccharides are not known. We aimed to identify the complete cDNAs of candidate genes implicated in SOS biosynthesis (B4GALT1, LALBA, ST3GAL5, ST6GAL1) and to analyse their expression during lactation in the Garganica and Maltese goat breeds. Moreover, we analysed the colostrum and milk contents of 3'-SL, 6'-SL and disialyllactose (DSL) and the possible correlations between expressed genes and SOS. RESULTS: We identified the complete coding cDNAs of B4GALT1 (HQ700335.1), ST3GAL5 (KF055858.2), and ST6GAL1 (HQ709167.1), the single nucleotide polymorphism (SNPs) of these genes and 2 splicing variants of the ST6GAL1 cDNA. RT-qPCR analysis showed that LALBA and ST6GAL1 were the genes with the highest and lowest expression in both breeds, respectively. The interaction effects of the breeds and sampling times were associated with higher levels of B4GALT1 and ST3GAL5 gene expression in Garganica than in Maltese goats at kidding. B4GALT1, LALBA, and ST3GAL5 gene expression changed from kidding to 60 and 120 days in Maltese goats, while in Garganica goats, a difference was observed only for the LALBA gene. Breed and lactation effects were also found for SOS contents. Positive correlations of B4GALT1, LALBA, ST3GAL5, and ST6GAL1 with 3'-SL/6'SL and DSL were found. CONCLUSIONS: The genetic effect on the oligosaccharide content of milk was previously highlighted in bovines, and this study is the first to investigate this effect in two goat breeds (Garganica and Maltese) during lactation. The genetic variability of candidate genes involved in SOS biosynthesis highlights their potential role in affecting gene expression and ultimately biological function. The investigation of gene regulatory regions as well as the examination of other sialyltransferase genes will be needed to identify the genetic pattern leading to a higher SOS content in the autochtonous Garganica breed and to protect it using a focused breeding strategy.


Asunto(s)
Calostro/química , Cabras/genética , Leche/química , Oligosacáridos/genética , Animales , Cruzamiento , ADN Complementario/análisis , Femenino , Perfilación de la Expresión Génica , Cabras/clasificación , Cabras/metabolismo , Lactancia , Oligosacáridos/biosíntesis , Polimorfismo de Nucleótido Simple , Embarazo
10.
Cell Physiol Biochem ; 46(5): 1768-1778, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29705805

RESUMEN

BACKGROUND/AIMS: Earlobe color is a typical external trait in chicken. There are some previous studies showing that the chicken white/red earlobe color is a polygenic and sex-linked trait in some breeds, but its molecular genetic and histological mechanisms still remain unclear. METHODS: We herein utilized histological section, genome-wide association study (GWAS) and RNA-seq, further to investigate the potential histological and molecular genetic mechanisms of white/red earlobe formation in Qiangyuan Partridge chicken (QYP). RESULTS: through histological section analysis, we found the dermal papillary layer of red earlobes had many more blood vessels than that of white earlobes. And we identified a total of 44 SNPs from Chromosome 1, 2, 3, 4, 9, 10, 11, 13, 19, 20, 23 and Z, that was significantly associated with the chicken white/red earlobe color from GWAS, along with 73 significantly associated genes obtained (e.g., PIK3CB, B4GALT1 and TP63), supporting the fact that the white/red earlobe color was also polygenic and sex-linked in QYP. Importantly, PIK3CB and B4GALT1 are both involved in the biological process of angiogenesis, which may directly give rise to the chicken white earlobe formation through regulating blood vessel density in chicken earlobe. Additionally, through contrast of RNA-seq profiles between white earlobe skins and red earlobe skins, we further identified TP63 and CDH1 differentially expressed. Combined with the existing knowledge of TP63 in epithelial development and tumor angiogenesis, we propose that down-regulated TP63 in white earlobes may play roles in thickening the skin and decreasing the vessel numbers in dermal papillary layer, thereby contributing to the white earlobe formation via paling the redness of the skin in QYP, but the specific mechanism remains to be further clarified. CONCLUSION: our findings advance the existing understanding of the white earlobe formation, as well as provide new clues to understand the molecular mechanism of chicken white/red earlobe color formation.


Asunto(s)
Pollos/genética , Animales , Pollos/anatomía & histología , Pollos/fisiología , Oído/anatomía & histología , Oído/fisiología , Femenino , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Masculino , Pigmentación
11.
BMC Cancer ; 18(1): 590, 2018 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-29793447

RESUMEN

BACKGROUND: The expression alterations of B4GALT1 have been noted in some types of cancer and they are related to cancer cell proliferation, invasiveness, metastasis, and drug resistance. We aimed to establish the expression of B4GALT1 in bladder cancer and its connection to patient outcomes, as well as forecasting the advantages of adjuvant chemotherapy (ACT) in patients with muscle-invasive bladder cancer (MIBC). METHODS: There were 142 and 112 MIBC patients who were consecutively recruited and treated via radical cystectomy from 2008 to 2012 in Shanghai Zhongshan Hospital and Fudan University Shanghai Cancer Center (FUSCC), respectively. Tissue microarrays (TMAs) were constructed in triplicate from specimens that had been fixed in formalin and embedded in paraffin samples. Immunohistochemistry was conducted to evaluate B4GALT1 expression in tumor cores, the connection between B4GALT1 expression and patients' clinical characteristics, and clinical results. RESULTS: B4GALT1 expression was not connected to clinical prognosis markers, but it was linked to overall survival (OS) (P = 0.013 and P = 0.010, respectively) in the two groups. Moreover, the high levels of B4GALT1 expression were independent indicators of poor OS (P = 0.026 and P = 0.046, respectively). Inclusion of B4GALT1 in the prognostic model revealed a greater predictive accuracy than the primary models. In addition, no differences were observed between B4GALT1 expression (low vs. high) and CD8+ T cell infiltration density (number/cm2) within tumor cores, but there was a positive Pearson correlation between B4GALT1 expression and expression of inhibitory receptor ligands, such as PD-L1 and CTLA4. Most significantly, the advantage of ACT noted in pT3/4 or N+ bladder cancer patients with low B4GALT1 expression was greater than in patients with a high B4GALT1 expression. CONCLUSIONS: Our evaluation indicated that B4GALT1 may be a possible prognosticator of MIBC, and it may be a predictive marker for the choice of ACT in pT3/4 or N+ patients.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Galactosiltransferasas/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Quimioterapia Adyuvante , China/epidemiología , Cistectomía , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Resultado del Tratamiento , Vejiga Urinaria/patología , Vejiga Urinaria/cirugía , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/cirugía
12.
Acta Biochim Biophys Sin (Shanghai) ; 48(12): 1120-1127, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27797721

RESUMEN

Zinc finger protein X-linked (ZFX) is a key regulator of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), which is required for both Notch intracellular domain (NotchIC)-induced acute T-cell leukemia and MLL-AF9-induced myeloid leukemia in mouse models. However, the role of ZFX and its underlying mechanism in human leukemic cells remain unclear yet, though accumulating data have demonstrated that ZFX is aberrantly expressed in various human tumors and plays an important role. Herein, we found that ZFX was aberrantly expressed in various human leukemic cell lines and primary cells from leukemia patients compared with control cells. The silence of ZFX led to the growth suppression through either the deregulated cell cycle or the induction of apoptosis in various cells including K562, Jurkat, Namalwa, and THP-1 cells. The gene expression analysis revealed that UDP-Gal:ßGlcNAc ß 1,4-galactosyltransferase, polypeptide 1 (B4GALT1) was significantly down-regulated upon ZFX silencing, which is implicated in the response of K562 cells to the treatment of imatinib mesylate (IM). In addition, lectin blot assay showed that the galactosylation of glycoproteins in K562 cells was suppressed upon ZFX silencing. Interestingly, overexpression of B4GALT1 restored the growth and conferred drug resistance to ZFX-silenced cells. Taken together, we have demonstrated that ZFX is aberrantly expressed in multiple human leukemic cells and it modulates the growth and drug response of leukemic cells partially via B4GALT1, which suggests that ZFX is a new regulator of leukemic cells and warrants intensive investigations on this 'stemness' regulator in these deadly diseases.


Asunto(s)
Galactosiltransferasas/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Leucemia de Células T/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Silenciador del Gen , Humanos , Factores de Transcripción de Tipo Kruppel/genética
13.
Cell Oncol (Dordr) ; 46(2): 283-297, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36454514

RESUMEN

PURPOSE: Recently, aberrant glycosylation has been recognized to be relate to malignant behaviors of cancer and outcomes of patients with various cancers. SLC35A2 plays an indispensable role on glycosylation as a nucleotide sugar transporter. However, effects of SLC35A2 on malignant behaviors of cancer cells and alteration of cancer cells surface glycosylation profiles are still not fully understood, particularly in hepatocellular carcinoma (HCC). Hence, from a glycosylation perspective, we investigated the effects of SLC35A2 on metastatic behaviors of HCC cells. METHODS: SLC35A2 expression in clinical samples and HCC cells was examined by immunohistochemical staining or Western blot/quantitative PCR and was regulated by RNA interference or vectors-mediated transfection. Effects of SLC35A2 expression alteration on metastatic behaviors and membrane glycan profile of HCC cells were observed by using respectively invasion, migration, cell adhesion assay, in vivo lung metastatic nude mouse model and lectins microarray. Co-location among proteins in HCC cells was observed by fluorescence microscope and detected by an in vitro co-immunoprecipitation assay. RESULTS: SLC35A2 was upregulated in HCC tissues, and is associated with poor prognosis of HCC patients. SLC35A2 expression alteration significantly affected the invasion, adhesion, metastasis and membrane glycan profile and led to the dysregulated expressions or glycosylation of cell adhesion-related molecules in HCC cells. Mechanistically, the maintenance of SLC35A2 activity is critical for the recruitment of the key galactosyltransferase B4GalT1, which is responsible for complex glycoconjugate and lactose biosynthesis, to Golgi apparatus in HCC cells. CONCLUSION: SLC35A2 plays important roles in promoting HCC metastasis by regulating cellular glycosylation modification and inducing the cell adhesive ability of HCC cells.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Transporte de Monosacáridos , Proteínas de Transporte de Nucleótidos , Animales , Humanos , Ratones , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Glicosilación , Neoplasias Hepáticas/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Transporte de Nucleótidos/metabolismo , Nucleótidos/metabolismo , Polisacáridos , Azúcares/metabolismo
14.
Protein Pept Lett ; 30(8): 668-678, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37198983

RESUMEN

BACKGROUND: UBE2Q1-dependent ubiquitination of key proteins including ß 1,4- galactosyltransferase (GalT1), and P53 might play a pivotal role in cancer development. OBJECTIVE: The present study aimed to evaluate the molecular analysis of possible interactions between UBE2Q1 with B4GALT1 and P53 proteins. METHODS: We established SW1116 colorectal cancer cell line stably transfected with UBE2Q1. To verify the overexpression of UBE2Q1, we performed western blot and fluorescent microscopy analysis. Using the immunoprecipitation (IP) product of the over-expressed protein on the silver staining gel, we observed the potential interacting partners of UBE2Q1. The Molecular Operating Environment (MOE) software was also used to perform the molecular docking of the UBC domain of UBE2Q1 (2QGX) with B4GALT1 (2AGD), and P53 (tetramerization (1AIE) and DNA binding domains (1GZH)) proteins. RESULTS: Western blot and IP analysis detected a UBE2Q1-GFP band in transfected cells, while no band was detected for mock-transfected cells. Moreover, the overexpression of UBE2Q1 tagged with GFP was observed under fluorescent microscopy as well with about 60-70% shining. Silver staining of IP gel revealed several bands in colorectal cancer (CRC) with UBE2Q1 overexpression. Protein- Protein interaction (PPI) analysis also depicted a high affinity of the UBC domain of UBE2Q1 to the B4GALT1 and P53 (tetramerization and DNA binding domains). Molecular docking also revealed hot-spot regions for all poses. CONCLUSION: Our data suggest that UBE2Q1 as an E2 enzyme of ubiquitination system can interact with B4GALT1 and P53, and may contribute to the accumulation of misfolded important proteins and colorectal tumor development.


Asunto(s)
Neoplasias Colorrectales , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Simulación del Acoplamiento Molecular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo
15.
Front Genet ; 13: 882004, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36568388

RESUMEN

Acute myeloid leukemia is the most prevalent type of leukemia in adults and is prone to relapse and chemoresistance, with a low long-term survival rate. Therefore, the identification of quality biomarkers constitutes an urgent unmet need. High expression of beta-1,4-galactosyltransferase 1 (B4GALT1) has been observed in several cancer types; however, its function in acute myeloid leukemia has rarely been studied. Therefore, our study obtained gene expression data from The Cancer Genome Atlas (TCGA) database to analyze the relationship between B4GALT1 and LAML. We compared the expression of B4GALT1 in LAML and healthy samples using the Wilcoxon rank-sum test. Furthermore, the association between B4GALT1 and survival rates was investigated using Kaplan-Meier analysis and Cox regression. The nomogram obtained by Cox analysis predicts the effect of B4GALT1 on the prognosis. To assess B4GALT1-related genes' enrichment pathway and function and the correlation between B4GALT1 and immune features, GO/KEGG, protein-protein interaction network, and single sample gene set enrichment analysis were used. In addition, B4GALT1-specific siRNAs were used to verify the effect of B4GALT1 on apoptosis. The results showed that B4GALT1 is overexpressed in LAML and has some reference value in the diagnostic and prognostic assessment of LAML. Moreover, functional enrichment showed that B4GALT1 and its 63 associated genes were closely associated with the negative regulation of the apoptotic signaling pathway. Silencing B4GALT1 significantly promoted apoptosis. In addition, B4GALT1 expression was positively correlated with the infiltration levels of macrophages, regulatory T-cell (Tregs), and Th17 cells; in contrast, B4GALT1 expression was negatively correlated with the infiltration levels of T helper cells, Mast cells, and NK cells. In conclusion, our study shows that B4GALT1 may play a vital role in the occurrence of LAML.

16.
Transl Cancer Res ; 11(3): 538-547, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35402178

RESUMEN

Background: Long noncoding RNAs (lncRNAs) are emerging as key players in the development and progression of cancer. Several malignancies involve dysregulated long noncoding ribonucleic acids (lncRNAs) in non-small cell lung cancer cell growth and their aggressive phenotypes. LncRNA B4GALT1-AS1 is important in the advancement of various malignancies, although its contribution to non-small cell lung cancer (NSCLC) remains unexplored. Methods: LncRNA B4GALT1-AS1 in NSCLC tissues was detected and further validated in a cohort of non-small cell lung cancer tissues. The effects of lncRNA B4GALT1-AS1 on proliferation were determined by in vitro experiments. The B4GALT1-AS1-miR-144-3p-ZEB1 axis was assessed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Furthermore, the mechanism of B4GALT1-AS1 was investigated using loss-of-function assays in vitro. Results: We showed significant upregulation of B4GALT1-AS1 in cell lines and tissues of NSCLC. B4GALT1-AS1 knockdown impeded the in vitro proliferation-related characteristics of the NSCLC cells. The demonstration of the binding capacity of B4GALT1-AS1 and miR-144-3p was predicted by bioinformatics and luciferase reporter activity assay. The B4GALT1-AS1 and miR-144-3p interaction was shown by using rescue experiments. NSCLC has a positive association with its target, zinc finger e-box binding homeobox 1 (ZEB1). Conclusions: In summary, the progression of NSCLC was facilitated by lncRNA B4GALT1-AS1 via interaction with miR-144-3p and positive regulation of ZEB1 expression.

17.
J Biotechnol ; 329: 92-103, 2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33549674

RESUMEN

Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.


Asunto(s)
Anticuerpos Monoclonales , Polisacáridos , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetinae , Cricetulus , Glicosilación
18.
Cancer Lett ; 500: 228-243, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33309857

RESUMEN

Aberrant glycosylation in pancreatic cancer has been linked to cancer development, progression and chemoresistance. However, the role of glycogene, such as galactosyltransferase, in pancreatic cancer remains unknown. Herein, we establish beta-1.4-galactosyltransferase 1 (B4GALT1) as a clinical marker and regulator of chemoresistance. Clinically, high B4GALT1 expression correlates with poor survival, enhanced tumor size, increased lymph node metastasis, elevated cancer progression and enhanced incidence of relapse in PDAC patients. Expression of B4GALT1 is up-regulated in gemcitabine resistant patient derived organoids as well as chemoresistant cancer cell lines, while genetic perturbation of its expression in PDAC cell lines regulates cancer progression and chemoresistance. Mechanistically, we show that elevated p65 activity transcriptionally up-regulates B4GALT1 expression, which then interacts with and stabilizes cyclin dependent kinase 11 isomer CDK11p110 protein via N-linked glycosylation, in order to promote cancer progression and chemoresistance. Finally, depletion of B4GALT1 rescues the response of chemoresistant cells to gemcitabine in an orthotopic PDAC model. Overall, our data uncovers a mechanism by which p65-B4GALT1-CDK11p110 signalling axis determines cancer progression and chemoresistance, providing a new therapeutic target for an improved pancreatic cancer treatment.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Quinasas Ciclina-Dependientes/genética , Galactosiltransferasas/genética , Factor de Transcripción ReIA/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicosilación/efectos de los fármacos , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Organoides/efectos de los fármacos , Gemcitabina
19.
Technol Cancer Res Treat ; 19: 1533033820980104, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33287670

RESUMEN

Our study was designed to investigate the role of B4GalT1 in glioblastoma, in vitro and in vivo, to detect whether B4GalT1 knockdown could regulate the development of glioblastoma, and further observe the relationship between B4GalT1 knockdown and the apoptosis and autophagy of glioblastoma. To begin, we looked at TCGA and GEPIA systems to predict the potential function of B4GalT1. Western blot and RT-PCR were used to analyze the expression, or mRNA level, of B4GalT1 at different tissue or cell lines. Next, the occurrence and development of glioblastoma, in vitro and in vivo, was observed by using B4GalT1 knocked down by lentivirus. Finally, the apoptosis and autophagy of glioblastoma was observed in vitro and in vivo. Results show that B4GalT1 was a highly variable gene, and GEPIA and TCGA systems show B4GalT1 expression in GBM tumor tissue was higher than in normal tissue. Pair-wise gene correlation analysis revealed a probable relationship between B4GalT1 and autophagy related proteins. The B4GalT1 expression and mRNA level were increased in tumor cells, or U87 cells. B4GalT1 knocked down by lentivirus could inhibit glioblastoma development, in vitro and in vivo, by reducing tumor weight and volume, increasing survival, and weakening tumor cells proliferation, migration, invasion. B4GalT1 knockdown could increase apoptosis and autophagy of glioblastoma in vitro and in vivo. Our study demonstrates that B4GalT1 may be able to regulate apoptosis and autophagy of glioblastoma. Bax, Bcl-2, cleaved caspase-3, Beclin-1, and LC3 s may be the downstream target factors of B4GalT1 in apoptosis and autophagy, which may provide a new strategy to reduce glioblastoma development by regulating apoptosis and autophagy.


Asunto(s)
Apoptosis/genética , Autofagia/genética , Galactosiltransferasas/genética , Glioblastoma/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Galactosiltransferasas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Modelos Biológicos , Ensayos Antitumor por Modelo de Xenoinjerto
20.
Oncol Lett ; 20(6): 284, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33014162

RESUMEN

Long non-coding (lnc) RNAs serve crucial functions in human cancers. However, the involvement of the lncRNA B4GALT1-antisense RNA 1 (AS1) in non-small cell lung cancer (NSCLC) has not been extensively studied. Reverse transcription-quantitative PCR was performed to detect B4GALT1-AS1 levels in NSCLC tissues and cell lines. Potential influences of B4GALT1-AS1 on biological functions of NSCLC were assessed through a series of in vitro experiments, and the molecular mechanism was determined via RNA immunoprecipitation (RIP) and bioinformatics analyses. The results of the present study demonstrated that knockdown of B4GALT1-AS1 significantly attenuated the proliferative ability and clonality of H1299 and A549 cells. In the present study, B4GALT1-AS1 competed as an endogenous RNA by sequestering microRNA-30e (miR-30e) leading to an enhanced expression of SRY-box transcription factor 9 (SOX9). The effects of silencing B4GALT1-AS1 on NSCLC cells proliferation could be ameliorated by inhibiting miR-30e or restoring SOX9. Hence, B4GALT1-AS1 acted as a lncRNA that drives tumor progression in NSCLC via the regulation of the miR-30e/SOX9 axis. The findings of the present study indicated that the B4GALT1-AS1/miR-30e/SOX9 axis maybe an effective target for NSCLC treatment and management.

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