An antibody for 6-sulfo sialyl Lewis x glycan

A group from Laboratory of Microbiology and Immunology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, etc. has developed an antibody for 6-sulfo sialyl Lewis x glycan.

To obtain an anti-6-sulfo sLex mAb, GlcNAc6ST-1/-2 DKO mice were immunized with Chinese hamster ovary (CHO) cells stably expressing 6-sulfo sLex that had been transiently transfected with an expression vector encoding Cmah.

The purified antibody named SF1 was subjected to glycan array analysis using CFG Glyucan microarray, to determine the glycan-binding specificity of SF1 in detail. It was confirmed that SF1 specifically bound to 6-sulfo sLex (Glycan #253) among all glycans on the array as follows.

Lymphocyte homing to peripheral lymph nodes is critically dependent on the interaction between the homing receptor, L-selectin, expressed on the surface of lymphocytes and 6-sulfo sialyl Lewis x expressed on the surface of specialized endothelial venules. Since SF1 significantly blocks lymphocyte homing, future studies using SF1 will advance our understanding of the role of 6-sulfo sLex in disease including rheumatoid arthritis, Sjögren syndrome, and ulcerative colitis.

Exosomes secreted from ST3G5-high cancer cells mediate peritoneal dissemination

A group from Department of Molecular Medicine and Biochemistry, Akita University Graduate School of Medicine, Akita, Japan, etc. has reported that lactosylceramide alpha-2,3-sialyltransferase (ST3G5) would be a suitable therapeutic target for preventing peritoneal dissemination mediated by exosomes secreted from ST3G5-high cancer cells.

In this paper, the roles of lactosylceramide alpha-2,3-sialyltransferase (ST3G5; also known as ST3GAL5 and GM3 synthase) in the exosome-mediated pre-metastatic niche in peritoneal milky spots (MSs)was investigated.

It was demonstrated that exosomes secreted from ST3G5-high cancer cells (ST3G5high-cExos) were found to contain high levels of h ypoxia-inducible factor 1-alpha (HIF1α) and glycolytic enzymes, and accumulated in MSs via uptake in macrophages owing to increased expression of sialic acid binding GM3 receptor (CD169; also known as Siglec1). The upreguration of CD169 further promotes the incorporation of exosomes via positive-feedback loop.
As is known, GM3 is the first molecule in ganglioside family biosynthesisformed by the transfer of sialic acid to lactosylceramide by ST3G5 and regulates cell adhesion, proliferation, and migration, and it was shown that HIF1α increases the expression of immune checkpoint molecules, PD-L1, and T cell exhaustion in MSs

This results suggest that ST3G5 would be a promising target for preventing peritoneal dissemination in some cancers.

Multi-omics evaluation was performed using a model in which Arabidopsis was inoculated with Trichoderma spp.

A group from Department for Sustainable Food Process, CRAST Research Centre, Università Cattolica del Sacro Cuore, Piacenza, Italy, etc. has reported about investigation of Trichoderma-mediated impact of heat, drought, and their combined stress on Arabidopsis thaliana, considering physiological responses, molecular implications at metabolome level, as well as the root and rhizosphere bacterial microbiota.

Plants treated with Trichodelma spp. were characterized by an increment of the fresh biomass even during drought and heat stress, suggesting that the increase in fresh weight observed was mainly due to an accumulation of water inside plant tissues.

The inoculation with Trichoderma spp. leaded to increased production of N-containing metabolites (including alkaloids and polyamines), phenylpropanoids, phytoalexins, tarpens, glucosinolates. It was also found that phytoholmons such as auxin-related substances (i.e., indole-3-acetaldehyde, indole-3-carboxaldehyde, and indole-3-ethanol), small peptides, and volatile organic compounds are upregulated by the inoculation with Trichoderma. And also, the soil microbiomes shifted in abundance and composition in response to environmental factors and Trichoderma treatments. Proteobacteria were the most predominant in roots and soil, on average, 89.6% of roots and 59.4 % of soil. Besides Proteobacteria in soil samples, one of the most abundant phyla were Bacteroides and Actinobacteroides.

They concluded that a holobiont approach, in other words, an multi-omics approach, is needed to understand the coordinated and complex dynamic interactions between the plant and its rhizosphere bacteria.

Concanavalin A (ConA) coated chitosan (CS) nanocarrier-DDS for antibiotic-resistant H. pylori

A group from Tissue Engineering and Regenerative Medicine Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran etc. has reported about a concanavalin A (ConA) coated chitosan (CS) nanocarrier-based drug delivery for the targeted release of peptides to the site of antibiotic-resistant H. pylori infection.

Helicobacter pylori is the cause of most cases of stomach ulcers and also causes some digestive cancers. The emergence and spread of antibiotic-resistant strains of H. pylori is one of the most important challenges in the treatment of its infections.

Chitosan was used as an encapsulating agent for CM11 peptide delivery by applying ionotropic gelation method. ConA was used for coating CS nanoparticles to target H. pylori. The size frequency for CS NPs and ConA-CS NPs was about 200 and 350 nm.

The Minimum Inhibitory Concentration (MIC) of the free CM11 peptide against drug-resistant H. pylori SS1 strain was 16 μg/ml while drug-resistant H. pylori SS1 showed MICs and MBCs 64 and >128 μg/ml against clarithromycin and amoxicillin, respectively. To encapsulate the peptide in nanoparticles, twice the MIC concentration of the peptide (32 µg/ml) was used.

CM11-loaded ConA-CS NPs has a higher antibacterial potential compared to the CM11-loaded CS NPs and free CM11 peptide, respectively. While there was no significant difference between the treatment with CM11-loaded ConA-CS NPs and triple antibiotics mixture, which indicates that it has the same effect similar to triple antibiotic therapy. CM11-loaded ConA-CS NPs and CM11-loaded CS NPs significantly reduced drug-resistant H. pylori SS1 after 12 h while amoxicillin and clarithromycin had no killing effect and their growth trend was same as the control group. The CM11-loaded ConA-CS NPs and triple antibiotics mixture were also able to kill drug-resistant H. pylori SS1 within 24 h while for free peptide and CM11-loaded CS NPs was after 48 h.

Changes in glycosylation of mitochondrial proteins due to Ischemia‐Reperfusion Injury using lectin microarrays

A group from Department of Cardiac and Pan-Vascular Diseases, Xi’an People’s Hospital (Xi’an Fourth Hospital), Xi’an, China, etc. has reported about changes in glycosylation of mitochondrial proteins due to Ischemia‐Reperfusion Injury using lectin microarrays.

Myocardial I/R injury seriously affects the recovery of the cardiac structure and function after cardiac surgery. I/R injury is a major obstacle in current clinical treatment, and its harm cannot be ignored. During severe myocardial ischaemia, the metabolism of myocardial cells is dominated by anaerobic glycolysis, which will cause the accumulation of acidic products and the depletion of ATP. After reperfusion, reactive oxygen species accumulation and Ca2+ overload are induced, leading to the opening of mitochondrial permeability transition pore (mPTP) which further leads to the disruption of the electron transport chain and the reduction of ATP production. As mPTP opens, matrix proteins and mitochondrial DNA are released into the cytoplasm. This process disrupts mitochondrial membrane potential and uncouples oxidative phosphorylation, leading to increased ATP consumption. Ca2+ overload and increased production of oxygen radicals will further activate inflammation and thrombosis, causing disruption of mitochondrial respiration, matrix swelling and mitochondrial membrane rupture, which leads to mitochondrial damage and cell death.

In this study, changes in glycosylation of mitochondrial proteins due to Ischemia‐Reperfusion Injury:I/RI were investigated by using lectin microarrays, and it was found that the expression of glycan structures recognized by LTL and SNA were significantly increased and the expression of glycan structures recognized by ECA were decreased with ischaemia for 45 min. Further analysis showed that the Siaα2-6Gal/GalNAc structure recognized by SNA were significantly increased.

About recent situations on Lectin microarrays and Glycan microarrays presented in Glyco26

Glyco26 was held at Academia Sinica in Taipei from August 27 to September 1, 2023.

First of all, I would like to summarize the trends in lectin arrays (also called lectin microarrays).
As you may know, lectin arrays appeared in 2005, and two groups, Lara Mahal from the United States and Jun Hirabayashi from Japan, published papers on glycan profiling analysis technology using lectin arrays. The following is one scene of Lara Mahal’s presentation (D010) from the University of Alberta at Glyco26.

Reference:A group of Lara Mahal et al.
Reference:A group of Jun Hirabayashi and Atsushi Kuno et al.
The latter paper adopted an evanescent-field fluorescence excitation method as the excitation technology to get a precise glycan profiling using a lectin array. This method enables detection of very weak biomolecular interactions between lectins immobilized on slide glasses and fluorescently labeled glycoproteins as analytes, non-destructively and directly from the liquid phase. And further, the admin. of this blog was also a co-author of this paper, and this technology was placed on the market as a glycan profiler (named GlycoStation® Reader 1200) in 2007 by myself.

Regarding the progress of researches using lectin arrays or on the lectin arrays themselves in Glyco26, regarding the former, there were presentations from a group of Academia Sinica and a group of AIST.
Wu-Show Su et al., Academia Sinica used lectin arrays for glycan profiling of IL6 secreted from cancer cells (A107), and Atsushi Kuno et al., AIST were verifying the differences in glycosylation at each local tissue site of cardic fibrosis using paraffin-embedded samples. In this study, an advanced glycan profiler, GSR2300 made by emukk LLC, was used, and it was shown that it is possible to get glycan profiling from only 3 cells (C050).
Regarding lectin arrays themselves, two groups gave presentations on arraying human lectins.
Kurt Drickamer et al., ICL develped an array immobilized with a total of 31 types of human lectins, including C-type lectins, Galectins, Siglec, and so forth to screen the binding specificity of these human lectins to pathogenic and commensal micro-organisms. This microarray provides unique information on how various lectins, which are at the forefront of human innate immunity, react with various microorganisms, and this demonstrates the usefulness of human lectin arrays (D026 ). In this study, human lectins were biotinylated and those were immobilized on a streptavidin-covered substrate. In addition, GlycoGenetics announced a microarray product that immobilizes 11 types of human lectins, mainly galectin (C072). Many of the existing lectin arrays are using plant lectins, such as LecChip, and lectin arrays adopting human lectins are a new trend in lectin arrays. However, the glycan binding specificity of human lectins is mainly related to the innate immune system, and therefore the coverage of glycans is not good enough. From the viewpoint of glycan profiling, existing lectin arrays centered on plant lectins are sufficiently powerful, and further they are highly comprehensive (for example, you can refer to arrays using plant lectins (C053) by Jaroslav Katrilik et al. of Slovak Academy of Sciences). So, existing lectin arrays and human lectin arrays will habitat segregation according to the applications, I think.

Next, regarding glycan arrays, as you know, the US CFG glycan arrays have become the de-facto position due to their high comprehensiveness. There were several presentations using this glycan array to evaluate the glycan binding specificity of glycan-binding proteins. One of the most interesting presentation at Glyco26 using glycan arrays would be “Blood Group A enhances SARS-CoV-2 infection” presented by Shang Chuen Wu, Richard Cummings and Sean Stowell et al., Harvard Medical School (A071). Ryuhei Hayashi and Yashuhiro Ozeki et al., Yokohama City Univ. used a unique glycan array (manufactured by GlycoTechnica) to evaluate the glycan binding specificity of marine sponge’s lectins . A newly discovered lectin named Chal18 was shown to have very strong specificity for T-antigen and to exhibited strong cytotoxicity against colorectal cancer cells (A031).
A new trend regarding glycan arrays is the movement to create arrays of glycans using pathogenic bacteria. Laurriel Macali and Todd Lowary et al., Academia Sinica was discussing the glycan binding specificity of mycobacteriophages using glycan arrays that immobilize 66 glycans exstracted from microorganisms. In this glycan array, glycans were immobilized on a slide glass using the neoglycoprotein method (i.e., a method in which a conjugate of an extracted glycan and BSA is made and the BSA is immobilized on the slide glass) (A040).
Microorganisms have a wide variety of glycans, and also their purification is difficult. Furthermore, since the state of glycans actually expressed on the surface of microorganisms and the state of extracted glycans and immobilized on glass slides are not the same, Hau-Ming Jan and Sean Stowell et al., Harvard Medical School proposed a method named MMA (microbe microarray)  in which the microorganisms are directly arrayed on a slide glass, and through studies on interaction of Gal-8 with glycan arrays and MMAs as an example, they concluded that MMA could predict binding properties more accurately than conventional glycan arrays (A048).

At Glyco26, there were a total of 343 presentations, so the admin. might not be able to cover them all and might have overlooked some. I appreciate your understanding about it and I hope this information is of some help to you.