Month: April 2022

A group from Department of Pulmonary Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan, etc. has reported about the difference in N-glycan modification of exosomes secreted from small-cell lung carcinomas and non-small-cell lung carcinomas.
https://www.jbc.org/article/S0021-9258(22)00390-8/fulltext

Lung cancer is a histologically complex disease that arises from different types of cells in the lungs. Small-cell lung carcinomas (SCLCs) and non-small-cell lung carcinomas (NSCLCs) are the two major lung cancer types classified based upon their histological properties. NSCLCs are further divided into three subtypes, lung adenocarcinomas (LUADs), squamous-cell lung carcinomas (SCCs), and large-cell lung carcinomas (LCCs), in descending order of frequency.

Compared lung cancers are as follows,
SCLC (H446 and SBC-3),
NSCLC-LUAD (HCC827),
NSCLC-SCC (H520, SK-MES-1, and LK-2), and
NSCLC-LCC (H1299).

Interestingly, the core N-glycan structures showed striking differences between SCLC-small extracellular vesicles(sEVs) and NSCLC-sEVs. NSCLC-sEVs were commonly enriched in bi-antennary and tri-antennary N-glycans, the core fucosylated forms being the major glycan structures. In contrast, the N-glycan profiles of SCLC-sEVs showed considerable heterogeneity, with an enrichment of N-glycans with antennal fucose (73% of total) in H446-sEVs and those with LacdiNAc (40% of total) or bisecting GlcNAc (29% of total) in SBC-3-sEVs.

As almost all N-glycans were sialylated, glycoproteins from the detergent-solubilized sEVs with SSA-conjugated beads and WGA-conjugated beads were pulled down, and the proteins were subjected to shotgun proteomics. The analysis revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs.

A group from Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France, etc. has found a β-trefoil lectin (R-type lectin) in protoctista, Salpingoeca rosetta.
https://www.biorxiv.org/content/10.1101/2022.02.10.479907v1.full

Lectin domains are often associated with other functional proteins such as enzymes or toxins. Life-threatening examples are ricin or cholera toxin, in which the lectin domain is responsible for the specificity and adhesion to cell surface glycans, prior to the cellular uptake of the toxin that interferes with metabolism.

Authors have found a eukaryotic Mytilec domain in the genome of Salpingoeca rosetta for the first time using the TrefLec database, and named it as SaroL-1. It has been kbnown that the Mytilec domain functions as a β-trefoil lectin (R-Type lectin), and binds efficiently to globotriaosyl ceramide (Gb3). β-trefoil lectins (R-type lectins) are widely distributed in bacteria, fungi, plants and animals.

The binding of SaroL-1 to different galactosyl-ligands was assessed in solution by isothermal titration calorimetry. The monosaccharides N-acetylgalactosamine (GalNAc) and α-methyl galactoside (GalαOMe) displayed a similar millimolar affinity with a Kd of 2.2 and 2.8 mM, respectively. All tested αGal disaccharides and the p-nitrophenyl-α-D-galactopyranoside (PNPG) derivative bound with affinities twice as strong, with a Kd close to 1 mM, except for αGal1-4Gal, the terminal disaccharide of the globoside Gb3. Lactose that contains βGal displayed very weak binding, being 20 times less efficient than αGal1-4Gal, confirming the preference for the αGal epitope.

SaroL-1 could form pore-like structures upon membrane binding and work as pore-forming toxins. Actually, SaroL-1 showed strong cytotoxicity towards cancer cells (H1299 cells). The dose-dependent increase of cytotoxicity was confirmed with a standard cell proliferation assay as shown below (a), and cell viability was reduced by approximately 87% after stimulation with SaroL-1. Interestingly, the soluble sugar PNPG could inhibit SaroL-1 cytotoxicity as shown below (b).

This lectin might be a valuable tool for cancer therapy.

A group from Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA, etc. has reported that virulence-modifying (VM) proteins are novel R-type lectins containing tandem N-terminal ricin B-chain-like β-trefoil domains
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9002632/

The so-called virulence-modifying (VM) proteins contributes to the pathogenesis of leptospirosis. Leptospirosis is a globally important neglected zoonotic disease.

From the homology searches of virulence-modifying (VM) proteins (PF07598), tandem ricin B-like lectin subdomains were identified in the amino–terminal regions of that protein. If so, ricin B chain, leptospiral VM proteins could bind to terminal galactose and N-acetyl-galactosamine residues, such as asialofetuin, as a model protein.
Actually, it was confirmed experimentally that the recombinant VM protein expressed in E. coli bound to asialofetuin, showing similar carbohydrate-binding specificity to ricin B chain as determined by competitive asialofetuin-binding assays.

Leptospira VM proteins bound to, and are quickly internalized by, HeLa cells via an N-terminal R-type lectin domain with specificity for terminal galactosyl residues. Cell surface binding and internalization were shown to be rapid, occurring within 30 min after exposure, and induced pleiotropic effects on HeLa cells, including actin depolymerization, caspase-3 activation, nuclear fragmentation, and ultimately blebbing and cell death.
One mechanism of cell death appears to be originated with genomic DNA degradation, which occurs after nuclear localization of the VM protein. Corroborating in vitro experiments using purified HeLa cell genomic DNA, and supercoiled and linearized bacterial plasmid DNA indicates that VM proteins tested so far, possess DNase activities. It was found that CTD of VM protein possess conserved active site residues identical to bovine DNase I.

So, the combination of cell surface binding, internalization, nuclear translocation, and DNase functions indicate that VM proteins evolved as novel forms of the bacterial AB domain-containing toxin paradigm.

A group from Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, etc. has reported that T-antigen (with or without sialylation) on MUC21 is required to elicit an anti-apoptotic effect.
https://pubmed.ncbi.nlm.nih.gov/35410995/

Mucins are highly O-glycosylated proteins thought to protect epithelial surfaces from physical, chemical, and biological insults, and attention to mucins has been paid in the fields of cancer diagnosis and cancer therapy. Mucin 21 (MUC21) is a unique transmembrane-type mucin, found in an endeavor to identify mouse epiglycanin, a mammary carcinoma-associated mucin.

It was first reported that expression of MUC21 by transfection makes epithelial cells resistant to apoptosis. For this effect to occur, MUC21’s O-glycans need to be extended to contain galactose (Gal) residues that may or may not be sialylated. In addition, we show that MUC21-dependent apoptosis resistance is not altered in the absence of galectin-3, suggesting that another galectin or carbohydrate-binding molecule is mediating the effect.

To investigate whether the extension of O-glycans with galactose is necessary for the apoptotic effect of MUC21, CHO-glycosylation variant ldlD cells were used. ldlD cells can synthesize O-glycans only after addition of GalNAc and/or galactose to the culture medium due to a deficiency in the enzyme UDP galactose and UDP-Gal/UDP-GalNAc 4-epimerase. ldlD–MUC21 transfectants grown in regular medium showed only one band at molecular weight around 160 kDa, indicating that ldlD cells expressed only nonglycosylated MUC21. When the medium was supplemented with GalNAc, bands at molecular weight around 200 kDa were seen when lysate was blotted with VVA lectin, suggesting that MUC21 carried Tn-antigen. When the medium was supplemented with GalNAc plus Gal, two bands appeared, one at approximately 200 kDa (no sialidase treatment) and one above 200 kDa (after sialidase treatment). The latter band was also seen when PNA lectin was used for blotting. These results indicate that GalNAc plus Gal supplementation led to the expression of sialyl T-antigen on MUC21.
Comparing these three kinds of samples, it was clearly shown that T-antigen (with or without sialylation) on MUC21 is required to elicit an anti-apoptotic effect.

Galectin-3 has been shown to have antiapoptotic effects in a variety of cell types. So, it was tested if galectin-3 is involved in the induction of MUC21-dependent apoptosis resistance by silencing galectin-3 in MUC21-transfectant HEK293 cells. However , it was found that galectin-3 silencing did not significantly alter the amount of apoptosis resistance in MUC21-transfectant cells.

What’s going on here?

A group from Department of Molecular Biology, Osaka International Cancer Institute, Chuo-ku, Osaka, Japan, etc. has reported that some urinary free-glycans were upregulated in some cancer patients.
https://pubmed.ncbi.nlm.nih.gov/35413075/

Free glycans in urine with α1,3-fucosylation, triply α2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core were upregulated in cancer patients. Simple α2,3-sialylation and α1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. The figure below shows such examples.

G: gastric cancer, P: pancreatic cancer, B: cholangiocarcinoma and C: colorectal cancer

A group from Institute of Medical Microbiology, Immunology and Parasitology Institute for Medical Microbiology, University Hospital Bonn, Bonn, Germany, etc. has reported about distinct N-Linked IgG glycosylation profile observed in Lymphatic filariasis.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8992374/

Lymphatic filariasis, commonly known as elephantiasis, is a tropical disease caused by vector-borne filarial parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori. In endemic areas, most infected individuals remain asymptomatic, showing no external signs of the infection while bearing microfilaria (MF⁺) and contributing to the transmission of the parasite. In contrast, putatively immune individuals, also known as endemic normals (EN), remain infection-free despite continuous exposition to the vector. In some of the exposed individuals, the infection develops into chronic pathology (CP), manifesting as lymphoedema (tissue swelling), elephantiasis (skin/tissue thickening) of limbs, or hydrocele (scrotal swelling).

The clinical outcomes of the infection are tightly linked to the host’s immune reactivity. Typically, alongside classical parasite-induced Th2 immune responses, asymptomatic patients present a strong immune-regulated profile with high levels of regulatory cells, anti-inflammatory cytokines.

The Fc glycans in the IgG molecule are bi-antennary glycans with varying fucose content, bisecting N-acetylglucosamine (GlcNAc), galactose (Gal), and sialic acid; most IgG molecules are fucosylated. These glycan structures have profound impacts on antibody functions and thereby on health. Agalactosylated and asialylated IgG glycoforms, for example, were seen to be particularly abundant in chronic inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (IBD), HIV, and mycobacterial infections.

Distinct N-Linked IgG glycosylation profiles observed are as follows:
CP patients presented significantly more glycan residues compared to MF⁺ and EN,
IgG from EN presented the highest numbers of agalactosylated residues,
IgG from CP patients presented the highest levels of both mono- and bigalactosylated residues,
IgG from MF⁺ patients presented lower Level of afucosylation, higher Level of sialylated residues, and bisecting GlcNAc.

This study has identified clear differences in IgG glycan profiles between CP, MF⁺ individuals, and EN and suggests that IgG glycome alterations might potentially be useful as biomarkers for disease severity prediction in lymphatic filariasis.

A group from Department of Transplantation, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico, etc. has reported about protective HLA alleles against COVID-19.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8982524/

In this pilot study in Tapachula, Chiapas México, the frequency of the allele HLA-A^*68 is diminished in patients with severe disease and who had a fatal outcome. HLA-A^*68 is an ancestral allele present in high frequencies in indigenous ancestral populations in Mexico, and the highest frequency is presented by the Teenek population, Yucatan Merida, and Yucatan rural inhabitants. The presence of protective alleles like HLA-A^{*68 could explain a little bit why this locality has been one of the least affected throughout Mexico, presenting low incidence rates of COVID-19.}

On the other hand, the HLA-A^*01 allele was associated with a fatal outcome.