Photosensitizer-thioglycosides enhance photodynamic therapy by augmenting cellular uptake.

Photodynamic therapy (PDT) uses photosensitizing agents along with light to ablate tissue, including cancers. Such light-driven localized delivery of free-radical oxygen to kill target tissue depends on photosensitizer cell penetration efficacy. While the attachment of monosaccharides and disaccharides to photosensitizers has been shown to potentially provide improved photosensitizer delivery, the range of glycan entities tested thus far is limited. We sought to expand such knowledge by coupling N-acetylglucosamine (GlcNAc) to pyropheophorbides as thioglycosides, and then testing photosensitizer efficacy. To this end, GlcNAc was conjugated to both pyropheophorbide-a and methyl pyropheophorbide-a. Among the entities tested, the conjugation of N-acetylglucosamine to methyl pyropheophorbide-a ('PSe') as thioglycoside enhanced cell uptake both in the presence and absence of human serum proteins, relative to other compounds tested. The enhanced PSe penetrance into cells resulted in higher cell death upon illumination with 665 nm light. While acting as a potent photosensitizer, PSe did not affect cellular carbohydrate profiles. Overall, the study presents a new pyropheophorbide glycoconjugate with strong in vitro PDT efficacy.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Knockout studies using CD34+ hematopoietic cells suggest that CD44 is a physiological human neutrophil E-selectin ligand.

The recruitment of peripheral blood neutrophils at sites of inflammation involves a multistep cascade, starting with E- and P-selectin expressed on the inflamed vascular endothelium binding sialofucosylated glycans on leukocytes. As the glycoconjugate biosynthesis pathways in different cells are distinct, the precise carbohydrate ligands of selectins varies both across species, and between different immune cell populations in a given species. To study this aspect in human neutrophils, we developed a protocol to perform CRISPR/Cas9 gene-editing on CD34+ hHSCs (human hematopoietic stem/progenitor cells) as they are differentiated towards neutrophil lineage. This protocol initially uses a cocktail of SCF (stem-cell factor), IL-3 (interleukin-3) and FLT-3L (FMS-like tyrosine kinase 3 ligand) to expand the stem/progenitor cells followed by directed differentiation to neutrophils using G-CSF (granulocyte colony-stimulating factor). Microfluidics based assays were performed on a confocal microscope platform to characterize the rolling phenotype of each edited cell type in mixed populations. These studies demonstrated that CD44, but not CD43, is a major E-selectin ligand on human neutrophils. The loss of function results were validated by developing sialofucosylated recombinant CD44. This glycosylated protein supported both robust E-selectin binding in a cell-free assay, and it competitively blocked neutrophil adhesion to E-selectin on inflamed endothelial cells. Together, the study establishes important methods to study human neutrophil biology and determines that sialoflucosylated-CD44 is a physiological human E-selectin ligand.

Cadherin-11 increases tumor cell proliferation and metastatic potential via Wnt pathway activation.

During epithelial-mesenchymal transition (EMT) in cancer progression, tumor cells switch cadherin profile from E-cadherin to cadherin-11 (CDH11), which is accompanied by increased invasiveness and metastatic activity. However, the mechanism through which CDH11 may affect tumor growth and metastasis remains elusive. Here, we report that CDH11 was highly expressed in multiple human tumors and was localized on the membrane, in the cytoplasm and, surprisingly, also in the nucleus. Interestingly, ß-catenin remained bound to carboxy-terminal fragments (CTFs) of CDH11, the products of CDH11 cleavage, and co-localized with CTFs in the nucleus in the majority of breast cancer samples. Binding of ß-catenin to CTFs preserved ß-catenin activity, whereas inhibiting CDH11 cleavage led to ß-catenin phosphorylation and diminished Wnt signaling, similar to CDH11 knockout. Our data elucidate a previously unknown role of CDH11, which serves to stabilize ß-catenin in the cytoplasm and facilitates its translocation to the nucleus, resulting in activation of Wnt signaling, with subsequent increased proliferation, migration and invasion potential.

A systems-based framework to computationally describe putative transcription factors and signaling pathways regulating glycan biosynthesis.

Glycosylation is a common posttranslational modification, and glycan biosynthesis is regulated by a set of glycogenes. The role of transcription factors (TFs) in regulating the glycogenes and related glycosylation pathways is largely unknown. In this work, we performed data mining of TF-glycogene relationships from the Cistrome Cancer database (DB), which integrates chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq data to constitute regulatory relationships. In total, we observed 22,654 potentially significant TF-glycogene relationships, which include interactions involving 526 unique TFs and 341 glycogenes that span 29 the Cancer Genome Atlas (TCGA) cancer types. Here, TF-glycogene interactions appeared in clusters or so-called communities, suggesting that changes in single TF expression during both health and disease may affect multiple carbohydrate structures. Upon applying the Fisher's exact test along with glycogene pathway classification, we identified TFs that may specifically regulate the biosynthesis of individual glycan types. Integration with Reactome DB knowledge provided an avenue to relate cell-signaling pathways to TFs and cellular glycosylation state. Whereas analysis results are presented for all 29 cancer types, specific focus is placed on human luminal and basal breast cancer disease progression. Overall, the article presents a computational approach to describe TF-glycogene relationships, the starting point for experimental system-wide validation.

Identification of cell-surface glycans that mediate motility-dependent binding and internalization of Pseudomonas aeruginosa by phagocytes.

Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP3), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1ß response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

B cells suppress medullary granulopoiesis by an extracellular glycosylation-dependent mechanism.

The immune response relies on the integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the marrow during emergency granulopoiesis but return upon restoration of homeostasis. Here we report a novel glycosylation-mediated crosstalk between marrow B cells and hematopoietic progenitors. Human B cells secrete active ST6GAL1 sialyltransferase that remodels progenitor cell surface glycans to suppress granulopoiesis. In mouse models, ST6GAL1 from B cells alters the sialylation profile of bone marrow populations, and mature IgD+ B cells were enriched in sialylated bone marrow niches. In clinical multiple myeloma, ST6GAL1 abundance in the multiple myeloma cells negatively correlated with neutrophil abundance. These observations highlight not only the ability of medullary B cells to influence blood cell production, but also the disruption to normal granulopoiesis by excessive ST6GAL1 in malignancy.

Selectins and Immune Cells in Acute Myocardial Infarction and Post-infarction Ventricular Remodeling: Pathophysiology and Novel Treatments.

The glycosciences aim to understand the impact of extracellular and intracellular carbohydrate structures on biological function. These glycans primarily fall into three major groups: lipid-linked carbohydrates that are referred to as glycosphingolipids or simply glycolipids; relatively short carbohydrate chains that are often O- or N-linked to proteins yielding common glycoproteins; and extended linear polymeric carbohydrate structures that are referred to as glycosaminoglycans (GAGs). Whereas, the impact of such carbohydrate structures has been extensively examined in cancer biology, their role in acute and chronic heart disease is less studied. In this context, a growing body of evidence indicates that glycans play an important role in immune mediated cell recruitment to damaged heart tissue to initiate wound healing and repair after injury. This is particularly important following ischemia and reperfusion that occurs in the heart in the setting of acute myocardial infarction. Here, immune system-mediated repair of the damaged myocardium plays a critical role in determining post-infarction ventricular remodeling, cardiac function, and patient outcome. Further, alterations in immune cell activity can promote the development of heart failure. The present review summarizes our current understanding of the phases of immune-mediated repair following myocardial infarction. It discusses what is known regarding glycans in mediating the recruitment of circulating immune cells during the early inflammatory stage of post-infarction repair, with focus on the selectin family of adhesion molecules. It offers future directions for research aimed at utilizing our knowledge of mechanisms underlying immune cell recruitment to either modulate leukocyte recruitment to the injured tissue or enhance the targeted delivery of biologic therapeutics such as stem cells in an attempt to promote repair of the damaged heart.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

The microRNA regulatory landscape of MSC-derived exosomes: a systems view.

Mesenchymal stem cell (MSC)-derived exosomes mediate tissue regeneration in a variety of diseases including ischemic heart injury, liver fibrosis, and cerebrovascular disease. Despite an increasing number of studies reporting the therapeutic effects of MSC exosomes, the underlying molecular mechanisms and their miRNA complement are poorly characterized. Here we microRNA (miRNA)-profiled MSC exosomes and conducted a network analysis to identify the dominant biological processes and pathways modulated by exosomal miRNAs. At a system level, miRNA-targeted genes were enriched for (cardio)vascular and angiogenesis processes in line with observed cardiovascular regenerative effects. Targeted pathways were related to Wnt signaling, pro-fibrotic signaling via TGF-ß and PDGF, proliferation, and apoptosis. When tested, MSC exosomes reduced collagen production by cardiac fibroblasts, protected cardiomyocytes from apoptosis, and increased angiogenesis in HUVECs. The intrinsic beneficial effects were further improved by virus-free enrichment of MSC exosomes with network-informed regenerative miRNAs capable of promoting angiogenesis and cardiomyocyte proliferation. The data presented here help define the miRNA landscape of MSC exosomes, establish their biological functions through network analyses at a system level, and provide a platform for modulating the overall phenotypic effects of exosomes.

A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis.

Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging because of the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MSn proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox; freely available from www.VirtualGlycome.org/glycopat). The program includes three major advances: (1) "SmallGlyPep," a minimal linear representation of glycopeptides for MSn data analysis. This format allows facile serial fragmentation of both the peptide backbone and PTM at one or more locations. (2) A novel scoring scheme based on calculation of the "Ensemble Score (ES)," a measure that scores and rank-orders MS/MS spectrum for N- and O-linked glycopeptides using cross-correlation and probability based analyses. (3) A false discovery rate (FDR) calculation scheme where decoy glycopeptides are created by simultaneously scrambling the amino acid sequence and by introducing artificial monosaccharides by perturbing the original sugar mass. Parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to catalogue site-specific glycosylation on simple glycoproteins, standard protein mixtures and human plasma cryoprecipitate samples in three common MS/MS fragmentation modes: CID, HCD and ETD. It is also used to identify 960 unique glycopeptides in cell lysates from prostate cancer cells. The results show that the simultaneous consideration of peptide and glycan fragmentation is necessary for high quality MSn spectrum annotation in CID and HCD fragmentation modes. Additionally, they confirm the suitability of GlycoPAT to analyze shotgun glycoproteomics data.

Bimodal Targeting Using Sulfonated, Mannosylated PEI for Combined Gene Delivery and Photodynamic Therapy.

Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer cells and tumor-associated macrophages (TAMs). Given the complex nature of tumor tissue, there could be merit in combining these strategies simultaneously. In this study, we developed a bimodal targeting approach to both cancer cells and macrophages, employing materials conducive to both gene delivery and PDT. Polymers libraries were created that consisted of cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide-a, with sulfonation (to target selectin-expressing cells) and mannosylation (to target TAMs). Polyplexes, consisting of these polymers electrostatically bound to DNA, were analyzed for transfection efficacy and cytotoxicity toward epithelial cells and macrophages to assess dual-targeting. This study provides preliminary proof of principle for using modified PEI for targeted gene delivery and PDT.

Current challenges for the targeted delivery and molecular imaging of stem cells in animal models.

In contrast to conventional, molecular medicine that focuses on targeting specific pathways, stem cell therapy aims to perturb many related mechanisms in order to derive therapeutic benefit. This emerging modality is inherently complex due to the variety of cell types that can be used, delivery approaches that need to be optimized in order to target the cellular therapeutic to specific sites in vivo, and non-invasive imaging methods that are needed to monitor cell fate. This review highlights advancements in the field, with focus on recent publications that use preclinical animal models for cardiovascular stem cell therapy. It highlights studies where cell adhesion engineering (CAE) has been used to functionalize stem cells to home them to sites of therapy, much like peripheral blood neutrophils. It also describes the current state of molecular imaging approaches that aim to non-invasively track the spatio-temporal pattern of stem cell delivery in living subjects.

Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion.

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 ß3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the ß1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galß1-4GlcNAcß1-6(Galß1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucß1-3 GalNAc and Fucα-1-2 D-Fucß-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc ß-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs): Pilot validation in porcine ischemia-reperfusion model.

Promising results are emerging in clinical trials focused on stem cell therapy for cardiology applications. However, the low homing and engraftment of the injected cells to target tissue continues to be a problem. Cellular glycoengineering can address this limitation by enabling the targeting of stem cells to sites of vascular injury/inflammation. Two such glycoengineering methods are presented here: i. The non-covalent incorporation of a P-selectin glycoprotein ligand-1 (PSGL-1) mimetic 19Fc[FUT7(+)] via lipid-protein G fusion intermediates that intercalate onto the cell surface, and ii. Over-expression of the α(1,3)fucosyltransferse FUT7 in cells. Results demonstrate the efficient coupling of 19Fc[FUT7(+)] onto both cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs), with coupling being more efficient when using protein G fused to single-tailed palmitic acid rather than double-tailed DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). This non-covalent cellular modification was mild since cell proliferation and stem-cell marker expression was unaltered. Whereas coupling using 19Fc[FUT7(+)] enhanced cell capture on recombinant P-selectin or CHO-P cell surfaces, α(1,3)fucosylation was necessary for robust binding to E-selectin and inflamed endothelial cells under shear. Pilot studies confirm the safety and homing efficacy of the modified stem cells to sites of ischemia-reperfusion in the porcine heart. Overall, glycoengineering with physiological selectin-ligands may enhance stem cell engraftment.

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13.

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.

Functionalization of cobalt porphyrin-phospholipid bilayers with his-tagged ligands and antigens.

Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can represent a substantial bottleneck in the formation of functionalized lipid-based materials. Although the polyhistidine tag (his-tag) has been transformative in its simplicity and efficacy in binding to immobilized metals, the successful application of this approach has been challenging in physiological settings. Here we show that lipid bilayers containing porphyrin-phospholipid conjugates that are chelated with cobalt, but not with other metals, can effectively capture his-tagged proteins and peptides. The binding follows a Co(II) to Co(III) transition and occurs within the sheltered hydrophobic bilayer, resulting in an essentially irreversible attachment in serum or in a million fold excess of competing imidazole. Using this approach we anchored homing peptides into the bilayer of preformed and cargo-loaded liposomes to enable tumour targeting without disrupting the bilayer integrity. As a further demonstration, a synthetic protein fragment derived from the human immunodeficiency virus was bound to immunogenic liposomes for potent antibody generation for an otherwise non-antigenic peptide.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.