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)1–2KKK 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.

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 9 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 eatme signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.

Hundreds of billions of cells undergo apoptosis in our body everyday and are removed by immunologically silent phagocytosis to maintain tissue homeostasis. Impairments in phagocytosis result in autoimmune and/or degenerative diseases. Eat-me signals are the key to the recognition of extracellular cargos and the initiation of the phagocytosis process by activating phagocytic receptors and signaling cascades, and are convenient targets for therapeutic modulation. Despite their importance, eat-me signals and other phagocytosis players are mostly identified on case-by-case basis with daunting challenges. This Commentary focuses on our latest knowledge of the extracellular players, highlights our approaches to systematically map unknown pathways by functional genetic and proteomic technologies, and discusses future direction to unravel the mystery of molecular phagocyte biology.

Despite many recent advances in genotype characterization of Enterocytozoon bieneusi worldwide and the exploration of the extent of cross-species transmission of microsporidiosis between humans and animals, the epidemiology of this neglected disease in China is poorly understood. In this study, a very high prevalence (60.3%; 94/156) of E. bieneusi infections in farmed pigs in Jilin province was detected by PCR of the ribosomal internal transcribed spacer (ITS). DNA sequence analysis of 88 E. bieneusi–positive specimens identified 12 distinct genotypes (11 known: CHN7, CS-1, CS-4, CS-6, EbpA, EbpB, EbpC, EbpD, EBITS3, G, and Henan-I; one novel: CS-9). Frequent appearance of mixed genotype infections was seen in the study animals. Weaned (74.6%; 53/71) or pre-weaned (68.8%; 22/32) pigs have infection rates significantly higher than growing pigs (35.8%; 19/53) (p<0.01). Likewise, E. bieneusi was detected in 2 of 45 sheep fecal specimens (4.4%) in Heilongjiang province, belonging to the known genotype BEB6. Genotypes EbpA, EbpC, EbpD, and Henan-I examined herein have been documented in the cases of human infections and BEB6, EbpA, EbpC, and EbpD in wastewater in central China. Infections of EbpA and EbpC in humans were also reported in other areas of the world. The other known genotypes (CHN7, CS-1, CS-4, CS-6, EBITS3, EbpB, and G) and the new genotype CS-9 were genetically clustered into a group of existing E. bieneusi genotypes with zoonotic potential. Thus, pigs could be a potential source of human E. bieneusi infections in China.