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International Journal of Molecular... Jun 2024The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as...
The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as abdominal pain, ultrasonography and altered laboratory parameters (C reactive protein, absolute neutrophil cell number and white blood cell number). The glycosylation pattern of serum N-glycome was analyzed in this study of 38 controls and 40 patients with pediatric appendicitis. The glycans were released by enzymatic deglycosylation followed by fluorescent labeling and solid-phase extraction. The prepared samples were analyzed by hydrophilic interaction liquid chromatography with fluorescence and mass-spectrometric detection. The generated data were analyzed by multiple statistical tests involving the most important laboratory parameters as well. Significant differences associated with the examined patient groups were revealed suggesting the potential use of glycosylation analysis supporting the detection of pediatric appendicitis.
Topics: Humans; Glycosylation; Appendicitis; Child; Male; Female; Adolescent; Polysaccharides; Biomarkers; Child, Preschool
PubMed: 38928139
DOI: 10.3390/ijms25126432 -
International Journal of Molecular... Jun 2024Observational studies revealed changes in Immunoglobulin G (IgG) N-glycosylation during the aging process. However, it lacks causal insights and remains unclear in which...
Observational studies revealed changes in Immunoglobulin G (IgG) N-glycosylation during the aging process. However, it lacks causal insights and remains unclear in which direction causal relationships exist. The two-sample bidirectional Mendelian randomization (MR) design was adopted to explore causal associations between IgG N-glycans and the senescence-associated secretory phenotype (SASP). Inverse variance weighted (IVW) and Wald ratio methods were used as the main analyses, supplemented by sensitivity analyses. Forward MR analyses revealed causal associations between the glycan peak (GP) and SASP, including GP6 (odds ratio [OR] = 0.428, 95% confidence interval [CI] = 0.189-0.969) and GP17 (OR = 0.709, 95%CI = 0.504-0.995) with growth/differentiation factor 15 (GDF15), GP19 with an advanced glycosylation end-product-specific receptor (RAGE) (OR = 2.142, 95% CI = 1.384-3.316), and GP15 with matrix metalloproteinase 2 (MMP2) (OR = 1.136, 95% CI =1.008-1.282). The reverse MR indicated that genetic liability to RAGE was associated with increased levels of GP17 (OR = 1.125, 95% CI = 1.003-1.261) and GP24 (OR = 1.222, 95% CI = 1.046-1.428), while pulmonary and activation-regulated chemokines (PARC) exhibited causal associations with GP10 (OR = 1.269, 95% CI = 1.048-1.537) and GP15 (OR = 1.297, 95% CI = 1.072-1.570). The findings provided suggested evidence on the bidirectional causality between IgG N-glycans and SASP, which might reveal potential regulatory mechanisms.
Topics: Humans; Mendelian Randomization Analysis; Glycosylation; Immunoglobulin G; Phenotype; Polysaccharides; Aging; Polymorphism, Single Nucleotide; Glycoproteins
PubMed: 38928043
DOI: 10.3390/ijms25126337 -
Biology Jun 2024The membrane glycoprotein CD133 (prominin-1) is widely regarded as the main molecular marker of cancer stem cells, which are the most malignant cell subpopulation within... (Review)
Review
The membrane glycoprotein CD133 (prominin-1) is widely regarded as the main molecular marker of cancer stem cells, which are the most malignant cell subpopulation within the tumor, responsible for tumor growth and metastasis. For this reason, CD133 is considered a promising prognostic biomarker and molecular target for antitumor therapy. Under normal conditions, CD133 is present on the cell membrane in glycosylated form. However, in malignancies, altered glycosylation apparently leads to changes in the functional activity of CD133 and the availability of some of its epitopes for antibodies. This review focuses on CD133's glycosylation in human cells and its impact on the function of this glycoprotein. The association of CD133 with proliferation, differentiation, apoptosis, autophagy, epithelial-mesenchymal transition, the organization of plasma membrane protrusions and extracellular trafficking is discussed. In this review, particular attention is paid to the influence of CD133's glycosylation on its immunodetection. A list of commercially available and custom antibodies with their characteristics is provided. The available data indicate that the development of CD133-based biomedical technologies should include an assessment of CD133's glycosylation in each tumor type.
PubMed: 38927329
DOI: 10.3390/biology13060449 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
Methods in Molecular Biology (Clifton,... 2024Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the...
Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.
Topics: CHO Cells; Cricetulus; Animals; CRISPR-Cas Systems; Gene Editing; Fucosyltransferases; Glycosylation; Fucose; Recombinant Proteins; Cricetinae; Humans
PubMed: 38926284
DOI: 10.1007/978-1-0716-3878-1_16 -
The Plant Journal : For Cell and... Jun 2024Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their...
Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.
PubMed: 38923617
DOI: 10.1111/tpj.16898 -
Virus Genes Jun 2024Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear...
Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.
PubMed: 38922563
DOI: 10.1007/s11262-024-02082-7 -
Toxins May 2024A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the...
A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the -glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin's origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants.
Topics: Ricin; Polysaccharides; Reference Standards; Protein Isoforms
PubMed: 38922138
DOI: 10.3390/toxins16060243 -
Current Issues in Molecular Biology Jun 2024We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant...
We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.
PubMed: 38921034
DOI: 10.3390/cimb46060363 -
Molecular Genetics and Metabolism Jun 2024Congenital disorders of glycosylation (CDG) are a continuously expanding group of monogenic disorders that disrupt glycoprotein and glycolipid biosynthesis, leading to...
INTRODUCTION
Congenital disorders of glycosylation (CDG) are a continuously expanding group of monogenic disorders that disrupt glycoprotein and glycolipid biosynthesis, leading to multi-systemic manifestations. These disorders are categorized into various groups depending on which part of the glycosylation process is impaired. The cardiac manifestations in CDG can significantly differ, not only across different types but also among individuals with the same genetic cause of CDG. Cardiomyopathy is an important phenotype in CDG. The clinical manifestations and progression of cardiomyopathy in CDG patients have not been well characterized. This study aims to delineate common patterns of cardiomyopathy across a range of genetic causes of CDG and to propose baseline screening and follow-up evaluation for this patient population.
METHODS
Patients with molecular confirmation of CDG who were enrolled in the prospective or memorial arms of the Frontiers in Congenital Disorders of Glycosylation Consortium (FCDGC) natural history study were ascertained for the presence of cardiomyopathy based on a retrospective review of their medical records. All patients were evaluated by clinical geneticists who are members of FCDGC at their respective academic centers. Patients were screened for cardiomyopathy, and detailed data were retrospectively collected. We analyzed their clinical and molecular history, imaging characteristics of cardiac involvement, type of cardiomyopathy, age at initial presentation of cardiomyopathy, additional cardiac features, the treatments administered, and their clinical outcomes.
RESULTS
Of the 305 patients with molecularly confirmed CDG participating in the FCDGC natural history study as of June 2023, 17 individuals, nine females and eight males, were identified with concurrent diagnoses of cardiomyopathy. Most of these patients were diagnosed with PMM2-CDG (n = 10). However, cardiomyopathy was also observed in other diagnoses, including PGM1-CDG (n = 3), ALG3-CDG (n = 1), DPM1-CDG (n = 1), DPAGT1-CDG (n = 1), and SSR4-CDG (n = 1). All PMM2-CDG patients were reported to have hypertrophic cardiomyopathy. Dilated cardiomyopathy was observed in three patients, two with PGM1-CDG and one with ALG3-CDG; left ventricular non-compaction cardiomyopathy was diagnosed in two patients, one with PGM1-CDG and one with DPAGT1-CDG; two patients, one with DPM1-CDG and one with SSR4-CDG, were diagnosed with non-ischemic cardiomyopathy. The estimated median age of diagnosis for cardiomyopathy was 5 months (range: prenatal-27 years). Cardiac improvement was observed in three patients with PMM2-CDG. Five patients showed a progressive course of cardiomyopathy, while the condition remained unchanged in eight individuals. Six patients demonstrated pericardial effusion, with three patients exhibiting cardiac tamponade. One patient with SSR4-CDG has been recently diagnosed with cardiomyopathy; thus, the progression of the disease is yet to be determined. One patient with PGM1-CDG underwent cardiac transplantation. Seven patients were deceased, including five with PMM2-CDG, one with DPAGT1-CDG, and one with ALG3-CDG. Two patients died of cardiac tamponade from pericardial effusion; for the remaining patients, cardiomyopathy was not necessarily the primary cause of death.
CONCLUSIONS
In this retrospective study, cardiomyopathy was identified in ∼6% of patients with CDG. Notably, the majority, including all those with PMM2-CDG, exhibited hypertrophic cardiomyopathy. Some cases did not show progression, yet pericardial effusions were commonly observed, especially in PMM2-CDG patients, occasionally escalating to life-threatening cardiac tamponade. It is recommended that clinicians managing CDG patients, particularly those with PMM2-CDG and PGM1-CDG, be vigilant of the cardiomyopathy risk and risk for potentially life-threatening pericardial effusions. Cardiac surveillance, including an echocardiogram and EKG, should be conducted at the time of diagnosis, annually throughout the first 5 years, followed by check-ups every 2-3 years if no concerns arise until adulthood. Subsequently, routine cardiac examinations every five years are advisable. Additionally, patients with diagnosed cardiomyopathy should receive ongoing cardiac care to ensure the effective management and monitoring of their condition. A prospective study will be required to determine the true prevalence of cardiomyopathy in CDG.
PubMed: 38917675
DOI: 10.1016/j.ymgme.2024.108513