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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 -
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 -
Frontiers in Immunology 2024This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1...
INTRODUCTION
This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells.
METHODS
Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both and . Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment.
RESULTS
Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by PET imaging and biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation.
DISCUSSION
This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.
Topics: Stomach Neoplasms; B7-H1 Antigen; Humans; Animals; Mice; Cell Line, Tumor; Glycosylation; Antibodies, Monoclonal, Humanized; Xenograft Model Antitumor Assays; Female; Positron-Emission Tomography
PubMed: 38915392
DOI: 10.3389/fimmu.2024.1405485 -
Acta Biochimica Et Biophysica Sinica Jun 2024FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic cells. Internal-tandem duplication domain (ITD) mutation and tyrosine kinase...
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic cells. Internal-tandem duplication domain (ITD) mutation and tyrosine kinase domain (TKD) mutation are the two most common mutations in acute myeloid leukemia (AML). Post-translational modifications (PTMs) of FLT3, such as glycosylation and ubiquitination, have been shown to impact various aspects of the protein in both wild-type (WT) and mutant forms of FLT3. In this review, we describe how the glycosylation status of FLT3 affects its subcellular localization, which significantly impacts the activation of downstream signaling, and the impact of specific ubiquitination on FLT3 function and stability, which may be associated with disease progression. Moreover, potential novel therapeutic strategies involving a combination of FLT3 tyrosine kinase inhibitors and drugs targeting glycosylation or ubiquitination are discussed.
PubMed: 38915288
DOI: 10.3724/abbs.2024112 -
[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate β-glucosyltransferase].Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent...
Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated -glycan moieties on peptide. It is of great significance to study the -glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues . In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate β-glucosyltransferase E (Alg5E) derived from . The results demonstrated that the recombinant Alg5E, expressed in , exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).
Topics: Glucosyltransferases; Substrate Specificity; Escherichia coli; Trichomonas vaginalis; Recombinant Proteins; Dolichol Phosphates; Endoplasmic Reticulum
PubMed: 38914494
DOI: 10.13345/j.cjb.230737 -
JCI Insight May 2024The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V...
The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.
Topics: Animals; Glycosylation; Mice; N-Acetylglucosaminyltransferases; Carcinoma, Pancreatic Ductal; Pancreatic Neoplasms; Cell Line, Tumor; Humans; T-Lymphocytes; Dendritic Cells; Killer Cells, Natural; Mice, Knockout
PubMed: 38912584
DOI: 10.1172/jci.insight.178804 -
Acta Biochimica Et Biophysica Sinica Jun 2024N-glycans play important roles in a variety of biological processes. In recent years, analytical technologies with high resolution and sensitivity have advanced...
N-glycans play important roles in a variety of biological processes. In recent years, analytical technologies with high resolution and sensitivity have advanced exponentially, enabling analysts to investigate N-glycomic changes in different states. Specific glycan and glycosylation signatures have been identified in multiple diseases, including cancer, autoimmune diseases, nervous system disorders, and metabolic and cardiovascular diseases. These glycans demonstrate comparable or superior indicating capability in disease diagnosis and prognosis over routine biomarkers. Moreover, synchronous glycan alterations concurrent with disease initiation and progression provide novel insights into pathogenetic mechanisms and potential treatment targets. This review elucidates the biological significance of N-glycans, compares the existing glycomic technologies, and delineates the clinical performance of N-glycans across a range of diseases.
PubMed: 38910518
DOI: 10.3724/abbs.2024101 -
Neuroscience Letters Jun 2024Brain somatic variants in SLC35A2, an intracellular UDP-galactose transporter, are commonly identified mutations associated with drug-resistant neocortical epilepsy and...
Brain somatic variants in SLC35A2, an intracellular UDP-galactose transporter, are commonly identified mutations associated with drug-resistant neocortical epilepsy and developmental brain malformations, including focal cortical dysplasia type I and mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). However, the causal effects of altered SLC35A2 function on cortical development remain untested. We hypothesized that focal Slc35a2 knockout (KO) or knockdown (KD) in the developing mouse cortex would disrupt cortical development and change network excitability. Through two independent studies, we used in utero electroporation (IUE) to introduce CRISPR/Cas9/targeted guide RNAs or short-hairpin RNAs into the embryonic mouse brain at day 14.5-15.5 to achieve Slc35a2 KO or KD, respectively, from neural precursor cells. Slc35a2 KO or KD caused disrupted radial migration of electroporated neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, importantly suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects of Slc35a2 mutations. Adult KO mice were implanted with EEG electrodes for 72-hour continuous recording. Spontaneous seizures were not observed in focal Slc35a2 KO mice, but there was reduced seizure threshold following pentylenetetrazol injection. Here we demonstrate that focal Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration and that KO leads to reduced seizure threshold. Together these results demonstrate a direct causal role for SLC35A2 in cortical development.
PubMed: 38909838
DOI: 10.1016/j.neulet.2024.137881