Lyar Is a New Ligand for Retinal Pigment Epithelial Phagocytosis.

Phagocytosis is critical to tissue homeostasis, as highlighted by phagocytosis defect of retinal pigment epithelial (RPE) cells with debris accumulation, photoreceptor degeneration and blindness. Phagocytosis ligands are the key to delineating molecular mechanisms and functional roles of phagocytes, but are traditionally identified in individual cases with technical challenges. We recently developed open reading frame phage display (OPD) for phagocytosis-based functional cloning (PFC) to identify unknown ligands. One of the identified ligands was Ly-1 antibody reactive clone (Lyar) with functions poorly defined. Herein, we characterized Lyar as a new ligand to stimulate RPE phagocytosis. In contrast to its reported nucleolar expression, immunohistochemistry showed that Lyar was highly expressed in photoreceptor outer segments (POSs) of the retina. Cytoplasmic Lyar was released from apoptotic cells, and selectively bound to shed POSs and apoptotic cells, but not healthy cells. POS vesicles engulfed through Lyar-dependent pathway were targeted to phagosomes and colocalized with phagosome marker Rab7. These results suggest that Lyar is a genuine RPE phagocytosis ligand, which in turn supports the validity of OPD/PFC as the only available approach for unbiased identification of phagocytosis ligands with broad applicability to various phagocytes.

Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis.

Tubby and tubby-like protein 1 (Tulp1) are newly identified phagocytosis ligands to facilitate retinal pigment epithelium (RPE) and macrophage phagocytosis. Both proteins without classical signal peptide have been demonstrated with unconventional secretion. Here, we characterized them as novel MerTK ligands to facilitate phagocytosis. Tulp1 interacts with Tyro3, Axl and MerTK of the TAM receptor tyrosine kinase subfamily, whereas tubby binds only to MerTK. Excessive soluble MerTK extracellular domain blocked tubby- or Tulp1-mediated phagocytosis. Both ligands induced MerTK activation with receptor phosphorylation and signalling cascade, including non-muscle myosin II redistribution and co-localization with phagosomes. Tubby and Tulp1 are bridging molecules with their N-terminal region as MerTK-binding domain and C-terminal region as phagocytosis prey-binding domain (PPBD). Five minimal phagocytic determinants (MPDs) of K/R(X)(1-2)KKK in Tulp1 N-terminus were defined as essential motifs for MerTK binding, receptor phosphorylation and phagocytosis. PPBD was mapped to the highly conserved 54 amino acids at the C-terminal end of tubby and Tulp1. These data suggest that tubby and Tulp1 are novel bridging molecules to facilitate phagocytosis through MerTK.

Galectin-3 is a new MerTK-specific eat-me signal.

Phagocytosis of apoptotic cells and cellular debris is a critical process of maintaining tissue and immune homeostasis. Defects in the phagocytosis process cause autoimmunity and degenerative diseases. Phagocytosis ligands or "eat-me" signals control the initiation of the process by linking apoptotic cells to receptors on phagocyte surface and triggering signaling cascades for cargo engulfment. Eat-me signals are traditionally identified on a case-by-case basis with challenges, and the identification of their cognate receptors is equally daunting. Here, we identified galectin-3 (Gal-3) as a new MerTK ligand by an advanced dual functional cloning strategy, in which phagocytosis-based functional cloning is combined with receptor-based affinity cloning to directly identify receptor-specific eat-me signal. Gal-3 interaction with MerTK was independently verified by co-immunoprecipitation. Functional analyses showed that Gal-3 stimulated the phagocytosis of apoptotic cells and cellular debris by macrophages and retinal pigment epithelial cells with MerTK activation and autophosphorylation. The Gal-3-mediated phagocytosis was blocked by excessive soluble MerTK extracellular domain and lactose. These results suggest that Gal-3 is a legitimate MerTK-specific eat-me signal. The strategy of dual functional cloning with applicability to other phagocytic receptors will facilitate unbiased identification of their unknown ligands and improve our capacity for therapeutic modulation of phagocytic activity and innate immune response.

Identification of Hnrph3 as an autoantigen for acute anterior uveitis.

Acute anterior uveitis (AAU) is the most common form of autoimmune uveitis in the eye with few known autoantigens. Identification of autoantigens will improve our understanding of the molecular mechanisms and capability for disease diagnosis. Phage display is a powerful technology for autoantigen identification. However, because of uncontrollable reading frames, phage display with conventional cDNA libraries identifies high percentage of non-open reading frames (non-ORFs) with minimal implications for autoantigen identification. We recently developed ORF phage display technology with minimal reading frame problem. Herein we used ORF phage display to identify 18 patient-specific clones, including 16 ORFs encoding endogenous proteins as candidate autoantigens for AAU. One of the identified antigens was heterogeneous nuclear ribonucleoprotein H3 (Hnrph3) that was further characterized for AAU relevance and independently verified by Western blot. These results demonstrate that ORF phage display is a valuable approach for identification of unknown autoantigens.

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display.

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1(-/-) mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.

Identification of calpain substrates by ORF phage display.

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions.

New perspective for phage display as an efficient and versatile technology of functional proteomics.

Phage display with antibody libraries has been widely used with versatile applications. However, phage display with cDNA libraries is rare and inefficient. Because of uncontrollable reading frames and stop codons in cDNA repertoires, high percentage of phage clones identified from conventional cDNA libraries are non-open reading frames (non-ORFs) encoding unnatural short peptides with minimal implications in protein networks. Consequently, phage display has not been used as a technology of functional proteomics to elucidate protein-protein interactions like yeast two-hybrid system and mass spectrometry-based technologies. Several strategies, including C-terminal display and ORF cDNA libraries, have been explored to circumvent the technical problem. The accumulative endeavors eventually led to the efficient elucidation of a large number of tubby- and phosphatidylserine-binding proteins in recent studies by ORF phage display with minimal reading frame issue. ORF phage display inherits all the versatile applications of antibody phage display, but enables efficient identification of real endogenous proteins with efficiency, sensitivity, and accuracy comparable to other technologies of functional proteomics. Its ELISA-like procedure can be conveniently adapted by individual laboratories or fully automated for high-throughput screening. Thus, ORF phage display is an efficient, sensitive, versatile, and convenient technology of functional proteomics for elucidation of global and pathway-specific protein-protein interactions, disease mechanisms, or therapeutic targets.

Efficient identification of phosphatidylserine-binding proteins by ORF phage display.

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in approximately 300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

Can phage display be used as a tool to functionally identify endogenous eat-me signals in phagocytosis?

Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis, and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to the understanding of phagocyte biology. Because of a lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. The feasibility of functional cloning of eat-me signals by phage display is investigated by characterizing the biological behavior of T7 phages displaying 2 well-known eat-me signals: growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). Gas6-phage binds to all 3 known Gas6 receptors: Mer, Axl, and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and nonphagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia, and retinal pigment epithelium cells, but not in nonphagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library.

Functional genome annotation through phylogenomic mapping.

Accurate determination of functional interactions among proteins at the genome level remains a challenge for genomic research. Here we introduce a genome-scale approach to functional protein annotation--phylogenomic mapping--that requires only sequence data, can be applied equally well to both finished and unfinished genomes, and can be extended beyond single genomes to annotate multiple genomes simultaneously. We have developed and applied it to more than 200 sequenced bacterial genomes. Proteins with similar evolutionary histories were grouped together, placed on a three dimensional map and visualized as a topographical landscape. The resulting phylogenomic maps display thousands of proteins clustered in mountains on the basis of coinheritance, a strong indicator of shared function. In addition to systematic computational validation, we have experimentally confirmed the ability of phylogenomic maps to predict both mutant phenotype and gene function in the delta proteobacterium Myxococcus xanthus.

The Myxococcus xanthus Nla4 protein is important for expression of stringent response-associated genes, ppGpp accumulation, and fruiting body development.

Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.

Secretogranin III as a disease-associated ligand for antiangiogenic therapy of diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of vision loss with retinal vascular leakage and/or neovascularization. Current antiangiogenic therapy against vascular endothelial growth factor (VEGF) has limited efficacy. In this study, we applied a new technology of comparative ligandomics to diabetic and control mice for the differential mapping of disease-related endothelial ligands. Secretogranin III (Scg3) was discovered as a novel disease-associated ligand with selective binding and angiogenic activity in diabetic but not healthy vessels. In contrast, VEGF bound to and induced angiogenesis in both diabetic and normal vasculature. Scg3 and VEGF signal through distinct receptor pathways. Importantly, Scg3-neutralizing antibodies alleviated retinal vascular leakage in diabetic mice with high efficacy. Furthermore, anti-Scg3 prevented retinal neovascularization in oxygen-induced retinopathy mice, a surrogate model for retinopathy of prematurity (ROP). ROP is the most common cause of vision impairment in children, with no approved drug therapy. These results suggest that Scg3 is a promising target for novel antiangiogenic therapy of DR and ROP.

Reticulocalbin-1 facilitates microglial phagocytosis.

Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes.

Hepatoma-derived growth factor-related protein-3 is a novel angiogenic factor.

Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.

Tubby regulates microglial phagocytosis through MerTK.

Immunologically-silent microglial phagocytosis of apoptotic cells and cellular debris is critical for CNS homeostasis and innate immune balance. The beneficial and detrimental effects of microglial phagocytosis on neurons remain controversial. Phagocytosis ligands are the key to selecting extracellular cargos, initiating the engulfment process, defining phagocyte functional roles and regulating phagocyte activities with therapeutic potentials. Here we characterized tubby as a new ligand to regulate microglial phagocytosis through MerTK receptor, which is well known for its immunosuppressive signaling. Tubby at 0.1nM significantly induced microglial phagocytosis of apoptotic cells with a maximal activity at 10nM. Tubby activated MerTK with receptor autophosphorylation in a similar dose range. Excessive soluble MerTK extracellular domain blocked tubby-mediated microglial phagocytosis of plasma membrane vesicles as cellular debris. Immunocytochemistry revealed that the ingested cargos were co-localized with MerTK-dependent non-muscle myosin II, whose rearrangement is necessary for cargo engulfment. Phagosome biomarker Rab7 was colocalized with cargos, suggesting that internalized cargos were targeted to phagocytic pathway. Tubby stimulated phagocytosis by neonatal and aged microglia with similar activities, but not by MerTK(-/-) microglia. These results suggest that tubby is a ligand to facilitate microglial phagocytosis through MerTK for the maintenance of CNS homeostasis.

Unconventional secretion of tubby and tubby-like protein 1.

Tubby-like proteins (Tulps) with no signal peptide have been characterized as cytoplasmic proteins with various intracellular functions, including binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. PI(4,5)P(2) has been implicated in unconventional secretion of fibroblast growth factor-2 without a signal peptide. Here, we show that all Tulps are expressed intracellularly and extracellularly. Tubby secretion is partially dependent on its PI(4,5)P(2)-binding activity with an essential secretory signal in the N-terminus. Pathogenic mutation in Tubby mice has no impact on tubby extracellular trafficking. Moreover, unconventional secretion of tubby and Tulp1 is independent of endoplasmic reticulum-Golgi pathway. These data implicate that Tulps may function extracellularly as well.

CbgA, a protein involved in cortex formation and stress resistance in Myxococcus xanthus spores.

CbgA plays a role in cortex formation and the acquisition of a subset of stress resistance properties in Myxococcus xanthus spores. The cbgA mutant produces spores with thin or no cortex layers, and these spores are more sensitive to heat and sodium dodecyl sulfate than their wild-type counterparts.

Nla18, a key regulatory protein required for normal growth and development of Myxococcus xanthus.

NtrC-like activators regulate the transcription of a wide variety of adaptive genes in bacteria. Previously, we demonstrated that a mutation in the ntrC-like activator gene nla18 causes defects in fruiting body development in Myxococcus xanthus. In this report, we describe the effect that nla18 inactivation has on gene expression patterns during development and vegetative growth. Gene expression in nla18 mutant cells is altered in the early stages of fruiting body development. Furthermore, nla18 mutant cells are defective for two of the earliest events in development, production of the intracellular starvation signal ppGpp and production of A-signal. Taken together, these results indicate that the developmental program in nla18 mutant cells goes awry very early. Inactivation of nla18 also causes a dramatic decrease in the vegetative growth rate of M. xanthus cells. DNA microarray analysis revealed that the vegetative expression patterns of more than 700 genes are altered in nla18 mutant cells. Genes coding for putative membrane and membrane-associated proteins are among the largest classes of genes whose expression is altered by nla18 inactivation. This result is supported by our findings that the profiles of membrane proteins isolated from vegetative nla18 mutant and wild-type cells are noticeably different. In addition to genes that code for putative membrane proteins, nla18 inactivation affects the expression of many genes that are likely to be important for protein synthesis and gene regulation. Our data are consistent with a model in which Nla18 controls vegetative growth and development by activating the expression of genes involved in gene regulation, translation, and membrane structure.