Lectin Domain Research Articles

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

BackgroundGrowing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear.MethodsA chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPβ-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro.ResultsSignificant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPβ, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPβ knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector.ConclusionsThis pre-clinical investigation reveals that C/EBPβ-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.

Beta-galactosidase gene family genome-wide identification and expression analysis of members related to fruit softening in melon (Cucumis melo L.)

BackgroundTexture quality is impotent for melon (Cucumis melo L.) fruit. β-galactosidase (β-Gal, EC 3.2.1.23) is an important cell wall glycosyl hydrolase involved in fruit softening, However, the β-Gal gene (BGALs) family hasn’t been identified genome-wide in melon. Thus, it’s necessary to conduct an in-depth bioinformatic analysis on melon BGALs family and to seek out the key members who participated in melon fruit softening.ResultsA total of 21 BGALs members designated as CmBGAL1-CmBGAL21 were identified genome-wide in melon, clustered into A-G seven clades. Among them, three duplications CmBGAL1:CmBGAL3, CmBGAL19:CmBGAL21, and CmBGAL20:CmBGAL21 happened. For conserved domains, besides the Glyco_hydro_35 domain (PF01301), all the members also contained the GHD domain (PF17834) except for CmBGAL12, and the Gal_Lectin (PF02140) domain existed in most CmBGALs at the C-termini. Motifs, protein secondary and tertiary structure analysis showed that the CmBGAL12 is a unique member. Moreover, protein-protein association network analysis showed that the CmBGAL12 is the only node protein. Furthermore, spatiotemporal expression pattern analysis by quantitative real-time PCR (qRT-PCR) suggested that most of CmBGALs expressed in tissues with vigorous cell wall remodeling/disassembly. In addition, cis-acting regulatory elements analysis in promoters inferred that CmBGALs might participate in diverse responsiveness to phytohormone, biotic and abiotic signaling.ConclusionsA novel clade of CmBGAL members (Clade F) related to melon fruit softening was discovered, since their expression showed a specific surge in the mature fruit of ‘HPM’ with mealy texture (softening sharply), but not in ‘HDB’ with crisp texture (softening bluntly). The homologous CmBGAL7–11 in Clade F exhibited identical spatiotemporal expression patterns may multiple genes leading to melon fruit softening.

Interference in melanoma CD248 function reduces vascular mimicry and metastasis

BackgroundTumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development.MethodsLoss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo.ResultsCD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis.ConclusionCD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.

Molecular characterization of a novel C-type lectin receptors (CD302) in Nile tilapia (Oreochromis niloticus) and its functional analysis in host defense against bacterial infection

C-type lectin receptors (CLRs) are a group of carbohydrate-binding proteins which function by mediating multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and the innate immune system's reaction to prospective invaders. However, how CLRs affects bony fish's innate immunity to bacterial infection is ill-defined. Therefore, CD302, a member of the CLRs family, was found in Nile tilapia (Oreochromis niloticus) (OnCD302) and the functions in the immune activities of tilapia against bacterial infection were examined. The OnCD302 consists of a C-type lectin domain, a transmembrane domain, and a deduced protein encoded by 242 amino acids. Besides, the OnCD302 protein has a series of highly conserved amino acid sites with other CD302 proteins. Subcellular localization showed that OnCD302 is located in the cell membrane. Transcriptional level investigation showed that OnCD302 was extensively expressed in all selected organs and had high expression in the liver. The transcriptional levels of OnCD302 are induced by Streptococcus agalactiae and Aeromonas hydrophila in the liver, spleen, head kidney, brain, and intestine. Afterwards, in vitro study revealed that several kinds of pathogens could be bound by the recombinant protein of OnCD302 (rOnCD302). Meanwhile, rOnCD302 could inhibit the growth of S. agalactiae, but had no inhibitory effect on A. hydrophila. However, the agglutinate ability of rOnCD302 to A. hydrophila and S. agalactiae was not observed in this study. Additionally, rOnCD302 reduced the ability of macrophages to phagocytose S. agalactiae and A. hydrophila. Summarily, our results indicated that CD302 get involved in fish immunization activities to bacterial infection.

Crystal structure of an L-type lectin domain from archaea.

The crystal structures of an L-type lectin domain from Methanocaldococcus jannaschii in apo and mannose-bound forms have been determined. A thorough investigation of L-type lectin domains from several organisms provides insight into the differences in these domains from different kingdoms of life. While the overall fold of the L-type lectin domain is conserved, differences in the lengths of the carbohydrate-binding loops and significant variations in the Mn2+ -binding site compared to the Ca2+ -binding site are observed. Furthermore, the sequence and phylogenetic analyses suggest that the archaeal L-type lectin domain is evolutionarily closer to the plant legume lectins than to its bacterial or animal counterparts. This is the first report of the biochemical, structural, sequence, and phylogenetic analyses of an L-type lectin domain from archaea and serves to enhance our understanding of the species-specific differences and evolution of L-type lectin domains.

The staphylococcal biofilm protein Aap mediates cell-cell adhesion through mechanically distinct homophilic and lectin interactions.

The accumulation phase of staphylococcal biofilms relies on both the production of an extracellular polysaccharide matrix and the expression of bacterial surface proteins. A prototypical example of such adhesive proteins is the long multidomain protein Aap (accumulation-associated protein) from Staphylococcus epidermidis, which mediates zinc-dependent homophilic interactions between Aap B-repeat regions through molecular forces that have not been investigated yet. Here, we unravel the remarkable mechanical strength of single Aap-Aap homophilic bonds between living bacteria and we demonstrate that intercellular adhesion also involves sugar binding through the lectin domain of the Aap A region. We find that the mechanical force needed to unfold individual β-sheet-rich G5-E domains from the Aap B-repeat regions is very high, ranging from 300 up to 1,000 pN at high loading rates, indicating these are extremely stable. This high mechanostability provides a means to the cells to form highly adhesive and cohesive biofilms capable of sustaining high physiological shear stress. Importantly, we identify a previously undescribed role of Aap in bacterial-bacterial adhesion, that is, heterophilic sugar binding by a specific lectin domain located in the N-terminal A region, which might be important to establish initial contacts between cells before strong homophilic bonds come into play. This study emphasizes the remarkable mechanical and binding properties of Aap as well as its wide diversity of adhesive functions.

Antimicrobial Studies on Garlic Lectin.

Allium sativum agglutinin (ASA) is an important lectin isolated from garlic bulbs and has shown promising therapeutic potential in earlier reports. It has a bulb-type lectin domain, and members of this protein family have been investigated for anti-cancer, antimicrobial and other effects. In our earlier study, we have reported ASA as an anti-cancer agent, and in the present study, we have evaluated it for its antifungal and antimicrobial effects. The effects of ASA on the opportunistic pathogens in humans Candida auris and Candida glabrata fungal strains have been evaluated, and efforts are made to evaluate the mechanistic basis of these antifungal effects. The antifungal activity of ASA on different strains of C. glabrata and C. auris was found with MIC50 concentration range of 30-70µg/ml. Fungal growth was significantly suppressed upon treatment with ASA at MIC50 and 2MIC50. Hydrogen peroxide production was detected after ASA treatment in fungal cells and cell morphology, and integrity was affected when analysed through FE-SEM. Further, the anti-biofilm effect of ASA was investigated against Candida and three bacterial strains (Escherichia coli, Staphylococcus aureus and Klebsiellapneumoniae), and promising results were obtained with maximal effect in case of K.pneumoniaeamong the bacterial strains. These results can form the basis for the development of ASA as antimicrobial agent.

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

Comparative transcriptomics reveals the immune dynamics during the molting cycle of swimming crab Portunus trituberculatus.

Molting is one of the most important biological processes of crustacean species, and a number of molecular mechanisms facilitate this complex procedure. However, the understanding of the immune mechanisms underlying crustacean molting cycle remains very limited. This study performed transcriptome sequencing in hemolymph and hepatopancreas of the swimming crab (Portunus trituberculatus) during the four molting stages: post-molt (AB), inter-molt (C), pre-molt (D), and ecdysis (E). The results showed that there were 78,572 unigenes that were obtained in the hemolymph and hepatopancreas of P. trituberculatus. Further analysis showed that 98 DEGs were involved in immunity response of hemolymph and hepatopancreas, and most of the DEGs participated in the process of signal transduction, pattern recognition proteins/receptors, and antioxidative enzymes system. Specifically, the key genes and pathway involved in signal transduction including the GPCR126, beta-integrin, integrin, three genes in mitogen-activated protein kinase (MAPK) signaling cascade (MAPKKK10, MAPKK4, and p38 MAPK), and four genes in Toll pathway (Toll-like receptor, cactus, pelle-like kinase, and NFIL3). For the pattern recognition proteins/receptors, the lowest expression level of 11 genes was found in the E stage, including C-type lectin receptor, C-type lectin domain family 6 member A and SRB3/C in the hemolymph, and hepatopancreatic lectin 4, C-type lectin, SRB, Down syndrome cell adhesion molecule homolog, Down syndrome cell adhesion molecule isoform, and A2M. Moreover, the expression level of copper/zinc superoxide dismutase isoform 4, glutathione peroxidase, glutathione S-transferase, peroxiredoxin, peroxiredoxin 6, and dual oxidase 2 in stage C or stage D significantly higher than that of stage E or stage AB. These results fill in the gap of the continuous transcriptional changes that are evident during the molting cycle of crab and further provided valuable information for elucidating the molecular mechanisms of immune regulation during the molting cycle of crab.

N-glycosylation of mannose receptor (CD206) regulates glycan binding by C-type lectin domains

The macrophage mannose receptor (MR, CD206) is a transmembrane endocytic lectin receptor, expressed in selected immune and endothelial cells, and is involved in immunity and maintaining homeostasis. Eight of the ten extracellular domains of the MR are C-type lectin domains (CTLDs) which mediate the binding of mannose, fucose, and GlcNAc in a calcium-dependent manner. Previous studies indicated that self-glycosylation of MR regulates its glycan binding. To further explore this structure-function relationship, we studied herein a recombinant version of mouse MR CTLD4-7 fused to human Fc-portion of IgG (MR-Fc). The construct was expressed in different glycosylation-mutant cell lines to study the influence of differential glycosylation on receptor glycan-binding properties. We conducted site-specific N- and O-glycosylation analysis and glycosylation site characterization using mass spectrometry by which several novel O-glycosylation sites were identified in mouse MR and confirmed in human full-length MR. This information guided experiments evaluating the receptor functionality by glycan microarray analysis in combination with glycan-modifying enzymes. Treatment of active MR-Fc with combinations of exoglycosidases, including neuraminidase and galactosidases, resulted in the loss of trans-binding (binding of MR CTLDs to non-MR glycans), due to unmasking of terminal, nonreducing GlcNAc in N-glycans of the MR CTLDs. Regalactosylation of N-glycans rescues mannose binding by MR-Fc. Our results indicate that glycans within the MR CTLDs act as a regulatory switch by masking and unmasking self-ligands, including terminal, nonreducing GlcNAc in N-glycans, which could control MR activity in a tissue- and cell-specific manner or which potentially affect bacterial pathogenesis in an immunomodulatory fashion.

A novel C-type lectin (CLEC12B) from Nile tilapia (Oreochromis niloticus) is involved in host defense against bacterial infection

C-type lectin (CLEC) is a family of carbohydrate-binding protein that has high affinity for calcium and mediates multiple biological events including adhesion between cells, the turnover of serum glycoproteins, and the innate immune system's reaction to prospective invaders. However, it's ill-defined for how CLEC effects bony fish's innate immunity to bacterial infection. Therefore, CLEC12B, a member of the C-type lectin domain family, was found in Nile tilapia (Oreochromis niloticus) and its functions in bacterial infection were examined. The OnCLEC12B consist of a C-type lectin domain, a transmembrane domain, and a hypothetical protein of 308 amino acids that encoded by 927 bp basic group. Besides, the OnCLEC12B protein have a series of highly conserved amino acid sites with other CLEC12B proteins. Subcellular localization showed that OnCLEC12B located in cell membrane. Transcriptional levels investigation showed that OnCLEC12B was extensively expressed in all selected organs and has high expression in the liver. The transcriptional levels of OnCLEC12B were induced by Streptococcus agalactiae and Aeromonas hydrophila in the liver, spleen, head kidney, brain, and intestine. Afterward, invitro study revealed that several kinds of pathogens could be bound and agglutinated by recombinant protein of OnCLEC12B (rOnCLEC12B). Moreover, rOnCLEC12B could not only promote the proliferation of monocytes/macrophages but also encourage its phagocytosis on S.agalactiae and A.hydrophila, and its over-expression could significantly suppress the activation of the NF-κB pathway. Summarily, our results indicated that OnCLEC12B gets involved in fish immunization activities to pathogens infection.

Purification, characterization and cDNA cloning of a lectin from the brittle star Ophioplocus japonicus.

Lectins are carbohydrate-binding proteins that possess specific sugar-binding properties and are involved in various biological activities in different organisms. In this study, purification, characterization, and cDNA cloning of a brittle star lectin, designated as Ophioplocus japonicus agglutinin (OJA), were conducted. OJA was isolated from the brittle star O. japonicus by affinity chromatography on a Sephadex G-25 column, followed by ion-exchange chromatography on a Resource Q column. This lectin yielded distinct bands at approximately 176 or 17kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing or reducing conditions, respectively. It also exhibited Ca2+-dependent hemagglutination activity, which, however, was not affected by other metal cations, such as Ba2+, Co2+, Cu2+, Zn2+, Fe2+, Mg2+, and Mn2+. The OJA activity was strongly inhibited by glucose and xylose among the monosaccharides tested, and by bovine thyroglobulin among the glycoproteins tested. Cloning of the OJA cDNA revealed that its primary structure contained the C-type lectin domain (CTLD). The results of this study showed that OJA is an echinoderm-derived glucose/xylose-specific lectin that belongs to the C-type lectin superfamily.

Pharmacokinetic evaluation of radiolabeled intraocular anti-CLEC14a antibody in preclinical animal species and application in humans.

Anti-angiogenic antibodies are widely used in the treatment of neovascular macular degeneration. Human antibody targeting C-type lectin domain family 14 member A (CLEC14a) is potential therapeutic agents owing to its antiangiogenic activity. In the present study, we aimed to predict the human intraocular pharmacokinetic (PK) properties of an anti-CLEC14a antibody. I-125 labeled aflibercept and anti-CLEC14a antibody were intravitreally injected into mice, rats, and rabbits. Single photon emission computed tomography/computed tomography imaging was performed, and the intraocular radioactivity concentration (%ID/ml) was obtained. The PK parameters in those three animal species were obtained by compartmental analysis. The PK parameters in humans were estimated by allometric scaling of the animal PK parameters with consideration of the hydrodynamic radius of the antibody. The mean half-life values of intraocular I-125-labeled aflibercept in mice, rats, and rabbits were 1.13 days, 1.25 days, and 4.91 days, respectively, by analysis with a one-compartment model. The predicted human half-life of intraocular aflibercept was 5.75 days based on vitreal volume by allometric scaling. The half-life values of intraocular I-125-labeled anti-CLEC14a in mice, rats and rabbits were 1.05 days, 1.84 days, and 6.37 days, respectively, by analysis with a one-compartment model. The predicted human half-life of intraocular anti-CLEC14a was 10.29 days based on vitreal volume. According to the hydrodynamic volume of the anti-CLEC14a, the predicted human half-life of intraocular anti-CLEC14a was 9.81 days. The PK characteristics of the intraocular anti-CLEC14a antibody were evaluated noninvasively in animals using I-125 labeling, and the intraocular PK characteristics in humans were predicted using these animal data. This methodology can be applied for the development of new antiangiogenic antibodies to treat macular degeneration.

240 Nutritional Intervention to Improve Carbohydrate Utilization and gut Health in Pigs

Abstract In swine production, using feed antibiotics as antimicrobial growth promotants has been reduced; thus, feed alternatives to manage gut health are required. Dietary fiber, resistant starch, oligosaccharides, and exopolysaccharides are carbohydrate structures and are nutritional tools that may enhance gut health in pigs. Dietary carbohydrates may have a role to alleviate diarrheal diseases including in pigs post-weaning but also reduce constipation in sows. Antibiotics are hypothesized to influence gut health via modulation of intestinal microbial profiles; fermentation and intestinal inflammation are considered important mechanisms. Dietary fiber sources differ in 2 key properties: fermentability and solubility. Rapid fermentation of fiber and oligosaccharides is associated with changes in microbial profiles and increased metabolite production. Recently, microbial composition was hypothesized to be less important and combined output of metabolites should be the focus. Fiber properties may also manipulate retention time and physico-chemical properties of the undigested residue; hence, non-fermentable insoluble fiber may prevent constipation in sows. Starch is mostly digested and absorbed as glucose. However, resistant starch is not digested and acts as insoluble but fermentable fiber and has unique properties, because it specifically increases intestinal abundance of bifidobacteria, which are associated with improved gut health. Using crop breeding, feed processing, and feed additives, the structure of fiber and starch can be altered thereby changing its degradation kinetics and affecting its utilization as energy source and its prebiotic activity. Finally, exopolysaccharides from Limosilactobacillus reuteri and unique oligosaccharides may serve as receptor analogues for pathogenic bacteria, e.g., enterotoxigenic Escherichia coli (ETEC). These receptor analogues block lectin domains of bacterial adhesins and thus prevent adherence of pathogens to the gut wall, thereby avoiding initiation of post-weaning diarrhea. In conclusion, dietary carbohydrates are important energy sources but may also provide important solutions to maintain gut health in pigs.

Poster session 1Roles of calreticulin and its mutants associated with MPN disorders in immune responses

MHC class I-β2m heterodimers (MHC-I) in the ER are typically loaded with peptides derived from cytosolic proteolysis, facilitated by the peptide-loading complex (PLC). Calreticulin (CRT) is an ER-localized lectin chaperone that is also an established member of the PLC. CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that also contains an ER-retention motif. Somatic frameshift CRT mutations (CRT-FS) have been shown to be associated with myeloproliferative neoplasms, a type of chronic, incurable blood tumor that can transform to acute myeloid leukemia. All mutations generate a common novel C-terminal domain that lacks the ER-retention signal and renders it positively charged. To study the effect of this mutation on antigen presentation by MHC-I, we first generated CRT-null HEK293T and found reduced surface MHC-I levels and association of MHC-I with low-affinity pep- tides. This phenotype was rescued by expression of CRT but not CRT-FS. CRT-FS was found to be secreted and no interaction with the PLC was detected, along with reduced recruitment of MHC-I. On retaining CRT-FS in the ER, it was found to associate with the PLC but did not restore MHC-I interaction with the PLC or surface MHC-I levels. Based on biochemical analysis and analysis of the PLC structure, we hypothesize that the altered C-terminal domain of CRT affects protein-protein interactions in the PLC that that render the complex ineffectual. Thus, the downregulation of MHC-I in CRT-FS-expressing tumor cells allows them to evade immune surveillance. We have used a patient-derived MPN cell line to generate cell lines lacking CRT expression, expressing CRT or CRT-FS, and have recapitulated the phenotypes reported above. These cell lines will be used to identify tumor-associated antigens and to investigate how extracellular CRT and CRT-FS influence innate immune responses, thus identifying potential immunotherapy targets.

Comparative Proteomic Analysis of Proteins in Breast Milk during Different Lactation Periods.

Breast milk is an unparalleled food for infants, as it can meet almost all of their nutritional needs. Breast milk in the first month is an important source of acquired immunity. However, breast milk protein may vary with the stage of lactation. Therefore, the aim of this study was to use a data-independent acquisition approach to determine the differences in the proteins of breast milk during different lactation periods. The study samples were colostrum (3–6 days), transitional milk (7–14 days), and mature milk (15–29 days). The results identified a total of 2085 different proteins, and colostrum contained the most characteristic proteins. Protein expression was affected by the lactation stage. The proteins expressed in breast milk changed greatly between day 3 and day 14 and gradually stabilized after 14 days. The expression levels of lactoferrin, immunoglobulin, and clusterin were the highest in colostrum. CTP synthase 1, C-type lectin domain family 19 member A, secretoglobin family 3A member 2, trefoil factor 3 (TFF3), and tenascin were also the highest in colostrum. This study provides further insights into the protein composition of breast milk and the necessary support for the design and production of infant formula.

Polypeptide N-acetylgalactosamine transferase 3: a post-translational writer on human health.

Polypeptide N-acetylgalactosamine transferase 3 (ppGalNAc-T3) is an enzyme involved in the initiation of O-GalNAc glycan biosynthesis. Acting as a writer of frequent post-translational modification (PTM) on human proteins, ppGalNAc-T3 has key functions in the homeostasis of human cells and tissues. We review the relevant roles of this molecule in the biosynthesis of O-GalNAc glycans, as well as in biological functions related to human physiological and pathological conditions. With main emphasis in ppGalNAc-T3, we draw attention to the different ways involved in the modulation of ppGalNAc-Ts enzymatic activity. In addition, we take notice on recent reports of ppGalNAc-T3 having different subcellular localizations, highlight critical intrinsic and extrinsic functions in cellular physiology that are exerted by ppGalNAc-T3-synthesized PTMs, and provide an update on several human pathologies associated with dysfunctional ppGalNAc-T3. Finally, we propose biotechnological tools as new therapeutic options for the treatment of pathologies related to altered ppGalNAc-T3. KEY MESSAGES: ppGalNAc-T3 is a key enzyme in the human O-GalNAc glycans biosynthesis. enzyme activity is regulated by PTMs, lectin domain and protein-protein interactions. ppGalNAc-T3 is located in human Golgi apparatus and cell nucleus. ppGalNAc-T3 has a central role in cell physiology as well as in several pathologies. Biotechnological tools for pathological management are proposed.

1723P CLEC3B mRNA expression levels are linked to distinct genetic backgrounds, transcriptomic signatures and survival in NSCLC

Downregulated C-type lectin domain family 3 member B (CLEC3B) is observed in NSCLC and is linked with immune cell infiltration. We aimed to analyse the impact of CLEC3B mRNA expression on survival and its molecular link to NSCLC subtypes and to immune cell infiltration. 19,892 samples were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes/WES), RNA (WTS) and IHC. Top quartile transcripts per million (TPM) were defined as high (Q4, ≥4.03 TPM) and bottom quartile as low (Q1, ≤0.89 TPM). Cell infiltration was estimated by QuantiSEQ. X2/Fisher-Exact were used and significance was determined as p-value adjusted for multiple comparison (q<0.05). Real-world overall survival (OS) information was obtained from insurance claims data. Median age was 69 years. High CLEC3B mRNA expression was associated with female sex (55.2% women vs 44.8% men, p<0.001). Adenocarcinomas vs squamous-cell carcinomas had higher CLEC3B expression (2.28 vs 1.51, q<0.001). CLEC3B correlated negatively with mutations in TP53 (74.7% in Q1 vs 52.4% in Q4), KEAP1 (15.9% vs 11.2%), RB1 (13.5 vs 9.2%), CDKN2A (13.0% vs 8.3%), NF1 (10.3% vs 6.9%) (all q<0.001). KRAS (31.8% in Q4 vs 25.6% in Q1), EGFR (17.2% vs 8.1%), STK11 (15.9% vs 11.3%) mutations were more frequently observed in CLEC3B high expressors (all q<0.001). High CLEC3B mRNA expression was negatively associated with high TMB (43.0% vs 29.0%) and high PD-L1 expression (62.5% vs 47.7%) (all q<0.05). Transcriptomics revealed an upregulation of various immunological markers (i.e. LAG3, INF-G, PDCD1) and a higher abundance of several immune cells (i.e. B cells, macrophages, T cells) in the CLEC3B high subgroup (all q<0.001). Patients with high CLEC3B mRNA expression showed an improved OS when compared to CLEC3B low expression (p<0.001, HR: 1.35); a similar observation was made in patients treated with checkpoint inhibitors (p<0.001, HR: 1.29). Our study represents the largest analysis of CLEC3B mRNA expression in NSCLC. High CLEC3B expression levels are linked to a distinct molecular/immunological profile and to improved survival. Further experiments are now ongoing to unravel functional aspects of CLEC3B biology in NSCLC.

CLEC5A is critical in Pseudomonas aeruginosa-induced NET formation and acute lung injury.

Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and it frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with LPS of P. aeruginosa and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa-induced pneumonia.