Gabriela Alvarado

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. J. Cell. Physiol. 227: 401–407, 2012.

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 ∼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. Copyright

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.

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.

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.

ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

Intracellular ABCF1 is identified and characterized as a new ligand to extrinsically stimulate retinal pigment epithelial cell phagocytosis. A new approach developed in this study is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to broaden understanding of extrinsic regulation and cargo recognition.

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.

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.

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. J. Cell. Biochem. 116: 2177–2187, 2015.