Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology.

METHODS: We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. RESULTS: Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. CONCLUSION: The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. RELEVANCE: The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

The TGF-ß-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-ß are well established, we wondered whether TGF-ß could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-ß promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-ß up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-ß type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-ß, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-ß signaling.

Virus entry. Lassa virus entry requires a trigger-induced receptor switch.

Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported 30 years ago to resist infection. We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.

Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide.

Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.

Cytotoxic CD4 T cells in antiviral immunity.

CD4 T cells that acquire cytotoxic phenotype and function have been repeatedly identified in humans, mice, and other species in response to many diverse pathogens. Since CD4 cytotoxic T cells are able to recognize antigenic determinants unique from those recognized by the parallel CD8 cytotoxic T cells, they can potentially contribute additional immune surveillance and direct effector function by lysing infected or malignant cells. Here, we briefly review much of what is known about the generation of cytotoxic CD4 T cells and describe our current understanding of their role in antiviral immunity. Furthering our understanding of the many roles of CD4 T cells during an anti-viral response is important for developing effective vaccine strategies that promote long-lasting protective immunity.

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay.

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.

COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes.

Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.

An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1.

The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment-cell-specific membrane glycoprotein, displaying features of G-protein-coupled receptors, yet exclusively localized to late endosomes, lysosomes and melanosomes. To dissect the signals responsible for the intracellular localization of OA1, we generated chimeric proteins consisting of the cytosolic domains of OA1 fused to the lumenal and transmembrane domains of LAMP1; in addition, we generated missense and deletion mutants of full-length OA1. Using this approach, we identified two separate sorting signals that are both necessary and sufficient for intracellular retention, as well as lysosomal and melanosomal localization, in melanocytic and non-melanocytic cells. These sorting signals are an unconventional dileucine motif within the third cytosolic loop and a novel motif, characterized by a tryptophan-glutamic acid doublet, within the C-terminal tail. Both motifs must be mutated to promote the plasma membrane localization of OA1, suggesting that they can independently drive its intracellular targeting. In addition, both motifs act similarly as lysosomal sorting signals in non-melanocytic cells, but appear to carry different specificities in melanocytic cells. Our findings indicate that OA1 contains multiple unconventional signals responsible for its lysosomal and melanosomal localization, and reveal a remarkable and unforeseen complexity in the regulation of polytopic protein sorting to specialized secretory organelles.