Transient Binding Dynamics of Complement System Pattern Recognition Molecules on Pathogens.

Previous studies of pattern recognition molecules (PRMs) of the complement system have revealed difficulties in observing binding on pathogens such as Aspergillus fumigatus and Escherichia coli, despite complement deposition indicative of classical and lectin pathway activation. Thus, we investigated the binding dynamics of PRMs of the complement system, specifically C1q of the classical pathway and mannose-binding lectin (MBL) of the lectin pathway. We observed consistently increasing deposition of essential complement components such as C4b, C3b, and the terminal complement complex on A. fumigatus and E. coli. However, C1q and MBL binding to the surface rapidly declined during incubation after just 2-4 min in 10% plasma. The detachment of C1q and MBL can be linked to complement cascade activation, as the PRMs remain bound in the absence of plasma. The dissociation and the fate of C1q and MBL seem to have different mechanistic functions. Notably, C1q dynamics were associated with local C1 complex activation. When C1s was inhibited in plasma, C1q binding not only remained high but further increased over time. In contrast, MBL binding was inversely correlated with total and early complement activation due to MBL binding being partially retained by complement inhibition. Results indicate that detached MBL might be able to functionally rebind to A. fumigatus. In conclusion, these results reveal a (to our knowledge) novel "hit-and-run" complement-dependent PRM dynamic mechanism on pathogens. These dynamics may have profound implications for host defense and may help increase the functionality and longevity of complement-dependent PRMs in circulation.

Serum Glycome as a Diagnostic and Prognostic Factor in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a risk factor for both mother and fetus/neonate during and after the pregnancy. Inconsistent protocols and cumbersome screening procedures warrant the search for new and easily accessible biomarkers. We investigated a potential of serum N-glycome to differentiate between healthy pregnant women (n = 49) and women with GDM (n = 53) using a lectin-based microarray and studied the correlation between the obtained data and parameters of glucose and lipid metabolism. Four out of 15 lectins used were able to detect the differences between the control and GDM groups in fucosylation, terminal galactose/N-acetylglucosamine (Gal/GlcNAc), presence of Galα1,4Galß1,4Glc (Gb3 antigen), and terminal α2,3-sialylation with AUC values above 60%. An increase in the Gb3 antigen and α2,3-sialylation correlated positively with GDM, whereas the amount of fucosylated glycans correlated negatively with the content of terminal Gal/GlcNAc. The content of GlcNAc oligomers correlated with the highest number of blood analytes, indices, and demographic characteristics, but failed to discriminate between the groups. The presence of terminal Gal residues correlated positively with the glucose levels and negatively with the LDL levels in the non-GDM group only. The results suggest fucosylation, terminal galactosylation, and the presence of Gb3 antigen as prediction markers of GDM.

Liquid Glycan Array.

The M13 phage platform is a stable and monodisperse nanoscale carrier, which can be modified with different molecules by chemical conjugation strategies. Here, we describe M13 phage acylated on pVIII protein with a dibenzocyclooctyne reacting with azido glycan to yield 30-1500 copy numbers of glycan per phage and monitored by MALDI-TOF spectrometry to generate multivalent glycoconjugates that contain desired densities of glycans. We prepared the liquid glycan arrays (LiGA) such that both the structure and density of glycans were encoded in the DNA of the bacteriophage. The LiGA can be used to validate the binding properties of glycans to purified lectins and explore the effect of glycan density on such binding. From a mixture of multivalent glycan probes, LiGAs can also identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and live animals in vivo.

Glycan-directed SARS-CoV-2 inhibition by leek extract and lectins with insights into the mode-of-action of Concanavalin A.

Four years after its outbreak, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global challenge for human health. At its surface, SARS-CoV-2 features numerous extensively glycosylated spike proteins. This glycan coat supports virion docking and entry into host cells and at the same time renders the virus less susceptible to neutralizing antibodies. Given the high genetic plasticity of SARS-CoV-2 and the rapid emergence of immune escape variants, targeting the glycan shield by carbohydrate-binding agents emerges as a promising strategy. However, the potential of carbohydrate-targeting reagents as viral inhibitors remains underexplored. Here, we tested seven plant-derived carbohydrate-binding proteins, called lectins, and one crude plant extract for their antiviral activity against SARS-CoV-2 in two types of human lung cells: A549 cells ectopically expressing the ACE2 receptor and Calu-3 cells. We identified three lectins and an Allium porrum (leek) extract inhibiting SARS-CoV-2 infection in both cell systems with selectivity indices (SI) ranging between >2 and >299. Amongst these, the lectin Concanavalin A (Con A) exerted the most potent and broad activity against a panel of SARS-CoV-2 variants. We used multiplex super-resolution microscopy to address lectin interactions with SARS-CoV-2 and its host cells. Notably, we discovered that Con A not only binds to SARS-CoV-2 virions and their host cells, but also causes SARS-CoV-2 aggregation. Thus, Con A exerts a dual mode-of-action comprising both, antiviral and virucidal, mechanisms. These results establish Con A and other plant lectins as candidates for COVID-19 prevention and basis for further drug development.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

Differential Glycoform Analysis of MUC1 Derived from Biological Specimens Using an Antibody-Overlay Lectin Microarray.

The association between altered glycosylation of MUC1 and various disease events has sparked significant interest. However, analytical technologies to investigate the disease-related glycoforms of endogenous MUC1 in blood and tissue specimens are limited. Therefore, we devised a reliable technique for differential analysis of endogenous MUC1 glycoforms based on an antibody-assisted lectin microarray. Its highly sensitive detection aids in analyzing soluble MUC1 from relatively small amounts of serum via a simple enrichment process. Micro-/macro-dissection of the MUC1-positive region is combined with glycoform analysis of the membrane-tethered MUC1. Thus, we have optimized the protocol for sample qualification using immunohistochemistry, sample pretreatment for tissue sections, protein extraction, purification via immunoprecipitation, and the antibody-overlay lectin microarray, which are sequentially essential for differential glycoform analysis of endogenous MUC1.

Analysis of the Interaction Between Mucin and Green Fluorescent Protein (GFP)-Tagged Galectin-2 Using a 96-Well Plate.

Due to a significant proportion of glycans binding to the peptide (constituting approximately 50-90% of the molecular weight), analyzing the interaction between the entire mucin molecule and its recognition protein (lectin) can be challenging. To address this, we propose a semiquantitative approach for measuring the interaction between mucin and lectin, which involves immobilizing mucin in a 96-well plate and subsequently adding lectin tagged with green fluorescent protein.

Structural and functional analysis of Cyanovirin-N homologs: Carbohydrate binding affinities and antiviral potential of cyanobacterial peptides.

Cyanobacteria, a group of photosynthetic prokaryotes, can sinthesize several substances due to their secondary metabolism, with notable properties, such as Cyanovirin-N(CVN), a carbohydrate-binding lectin, that exhibits antiviral activity against several pathogens, due to its ability to bind viral surface carbohydrates such as mannose, thus interfering with the viral entry on the cell. CVN has been described in several cyanobacterial strains and shows biotechnological potential for the development of drugs of pharmaceutical interest. This study focuses on the genomic exploration and characterization of Cyanovirin-N homologs to assess the conservation of carbohydrate-binding affinity within the group. The analysis of their antiviral properties was carried out using bioinformatics tools to study protein models through an in silico pipeline, following the steps of genomic prospection on public databases, homology modeling, docking, molecular dynamics and energetic analysis. Mannose served as the reference ligand, and the lectins' binding affinity with mannose was assessed across Cyanovirin-N homologs. Genomic mining identified 33 cyanobacterial lectin sequences, which underwent structural and functional characterization. The results obtained from this work indicate strong carbohydrate affinity on several homologs, pointing to the conservation of antiviral properties alongside the group. However, this affinity was not uniformly distributed among sequences, exhibiting significant heterogeneity in binding site residues, suggesting potential multi-ligand binding capabilities on the Cyanovirin-N homologs group. Studies focused on the properties involved in these molecules and the investigation of the genetic diversity of Cyanovirin-N homologs could provide valuable insights into the discovery of new drug candidates, harvesting the potential of bioinformatics for large-scale functional and structural analysis.

Glycan-Lectin interactions between platelets and tumor cells drive hematogenous metastasis.

Glycosylation is a ubiquitous cellular or microenvironment-specific post-translational modification that occurs on the surface of normal cells and tumor cells. Tumor cell-associated glycosylation is involved in hematogenous metastasis. A wide variety of tumors undergo aberrant glycosylation to interact with platelets. As platelets have many opportunities to engage circulating tumor cells, they represent an important avenue into understanding the role glycosylation plays in tumor metastasis. Platelet involvement in tumor metastasis is evidenced by observations that platelets protect tumor cells from damaging shear forces and immune system attack, aid metastasis through the endothelium at specific sites, and facilitate tumor survival and colonization. During platelet-tumor-cell interactions, many opportunities for glycan-ligand binding emerge. This review integrates the latest information about glycans, their ligands, and how they mediate platelet-tumor interactions. We also discuss adaptive changes that tumors undergo upon glycan-lectin binding and the impact glycans have on targeted therapeutic strategies for treating tumors in clinical settings.

Tumor hematogenous metastasis is a serious threat to the survival and prognosis of patients, and a variety of factors help this process to occur, and platelets are also involved. During tumor cell metastasis, platelets can adhere to each other and tumor cells, a phenomenon that leads to the immunity of tumor cells from various threats in metastasis, including immune attacks, shearing forces, etc. Scientists have shown that the adhesion effect between platelets and tumor cells is often dependent on various types of sugars, which are not the sugars we ingest. These sugars often appear as glycosylation modifications on the proteins of the cells, including normal glycosylation modifications and some abnormal structures that only appear on tumor cells, and their ligands, lectins, are also present on the surface of the tumor cells or platelets. Their combination results in the better adaptation of tumor cells to the metastatic process, where proteins such as P-selectin, CLEC-2, and Galectins have been more studied. Focusing on Glycan-Lectin interactions between platelets and tumor cells, related studies help us to further understand tumor metastasis, and intervene in this binding and develop related drugs with great potential.

The Lectin Pathway of the Complement System-Activation, Regulation, Disease Connections and Interplay with Other (Proteolytic) Systems.

The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation-fibrinolytic system. Among the three main activation routes of complement, the lectin pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary ("moonlighting") functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components.

het-B allorecognition in Podospora anserina is determined by pseudo-allelic interaction of genes encoding a HET and lectin fold domain protein and a PII-like protein.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species.

Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors.

In vivo analysis of protein function in nociceptor subpopulations using antisense oligonucleotides and short interfering RNAs is limited by their non-selective cellular uptake. To address the need for selective transfection methods, we covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors. Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA were found to have: (a) less PKCε in dorsal root ganglia (DRG), (b) reduced PKCε expression in IB4(+) but not IB4(-) DRG neurons, and (c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)-nociceptors. These results establish that IB4-streptavidin can be used to study protein function in a defined subpopulation of nociceptive C-fiber afferents.

The impact of glycosylation on the structure, function, and interactions of CD14.

CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. ßGal, α2,3 and α2,6 sialylated ßGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific.

Mannose-specific plant and microbial lectins as antiviral agents: A review.

Lectins are non-immunological carbohydrate-binding proteins classified on the basis of their structure, origin, and sugar specificity. The binding specificity of such proteins with the surface glycan moiety determines their activity and clinical applications. Thus, lectins hold great potential as diagnostic and drug discovery agents and as novel biopharmaceutical products. In recent years, significant advancements have been made in understanding plant and microbial lectins as therapeutic agents against various viral diseases. Among them, mannose-specific lectins have being proven as promising antiviral agents against a variety of viruses, such as HIV, Influenza, Herpes, Ebola, Hepatitis, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and most recent Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The binding of mannose-binding lectins (MBLs) from plants and microbes to high-mannose containing N-glycans (which may be simple or complex) of glycoproteins found on the surface of viruses has been found to be highly specific and mainly responsible for their antiviral activity. MBLs target various steps in the viral life cycle, including viral attachment, entry and replication. The present review discusses the brief classification and structure of lectins along with antiviral activity of various mannose-specific lectins from plants and microbial sources and their diagnostic and therapeutic applications against viral diseases.

Altered O-linked glycosylation in benign and malignant meningiomas.

Background: Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Methods: Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Results: Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 ß-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Conclusion: Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Synthetic assembly of α-O-linked-type GlcNAc using polymer chemistry affords sugar clusters, which effectively bind to lectins.

Fischer's glycoside synthesis was applied to linker precursor alcohols of two different lengths having appropriate alkane chains to obtain the corresponding α-glycoside and it was found to be applicable with moderate yields. Water-soluble glycomonomers were systematically prepared from N-acetyl-d-glucosamine (GlcNAc) by introducing two kinds of alcohols having different methylene lengths. Typical radical polymerizations of the glycomonomers with acrylamide as a modulator for control of the distance between carbohydrate residues in water in the presence of ammonium persulfate (APS)-N,N,N',N'-tetramethylethylenediamine (TEMED) gave a series of glycopolymers with various α-glycoside-type GlcNAc residue densities. Fluorometric analysis of the interaction of wheat germ agglutinin (WGA) with the glycopolymers was performed and the results showed unique binding specificities based on structural differences.

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility.

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.