Lijun Feng

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes

Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease affecting the related subcellular organelles platelet dense granules, lysosomes, and melanosomes. The mouse genes for HPS, pale ear and pearl, orthologous to the human HPS1 and HPS2 (ADTB3A) genes, encode a novel protein of unknown function and the beta(3)A subunit of the AP-3 adaptor complex, respectively. To test for in vivo interactions between these genes in the production and function of intracellular organelles, mice doubly homozygous for the 2 mutant genes were produced by appropriate breeding. Cooperation between the 2 genes in melanosome production was evident in increased hypopigmentation of the coat together with dramatic quantitative and qualitative alterations of melanosomes of the retinal pigment epithelium and choroid of double mutant mice. Lysosomal and platelet dense granule abnormalities, including hyposecretion of lysosomal enzymes from kidneys and depression of serotonin concentrations of platelet dense granules were likewise more severe in double than single mutants. Also, lysosomal enzyme concentrations were significantly increased in lungs of double mutant mice. Interaction between the 2 genes was specific in that effects on organelles were confined to melanosomes, lysosomes, and platelet dense granules. Together, the evidence indicates these 2 HPS genes function largely independently at the whole organism level to affect the production and function of all 3 organelles. Further, the increased lysosomal enzyme levels in lung of double mutant mice suggest a cause of a major clinical problem of HPS, lung fibrosis. Finally, doubly mutant HPS mice are a useful laboratory model for analysis of severe HPS phenotypes.

Lumenal protein multimerization in the distal secretory pathway/secretory granules.

Differences in protein solubility appear to play an important role in lumenal protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric protein assembly is one of the factors regulating the efficiency of protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.

Comparative genomic and phylogenetic analyses of the intelectin gene family: implications for their origin and evolution.

Intelectin is a newly characterized gene family involved in early embryogenesis, host-pathogen interactions and iron metabolism. In this study, we searched the genomes of metazoans by extensive BLAST survey and found no intelectin homologs in invertebrate metazoans but 12 in amphioxus Branchiostoma floridae and 21 in ascidians Ciona intestinalis. Some ascidians oocyte cortical granule lectins (CGLs) have unknown insertion sequences between fibrinogen-related domain (FReD) and Intelectin Domain, the boundaries of which are equivalent to exon structures. In addition to ascidians intelectins/CGLs located in the base, phylogenetic tree comprises four main clades representing mammal, frog, fish, and amphioxus, indicating that intelectin genes undergo extensive lineage-specific duplication or gene conversion. However, genomic neighborhood surrounding analysis shows that clear proto-orthologies are difficult to be established among these counterparts. In addition, sequence comparison and phylogenetic analysis of FReDs from intelectins and other fibrinogen-like proteins from choanoflagellate, anemone, frog and human indicate FReDs of intelectins are unique. Likewise, these choanoflagellate and anemone genes may be close to intelectin gene.

Identification and primary immune characteristics of an amphioxus akirin homolog

Akirin is a recently described nuclear protein that is thought to be required for the NF-κB signaling pathway in insects and vertebrates. Here, functional investigations of akirin are described in the basal chordate amphioxus Branchiostoma belcheri tsingtauense in an attempt to link this gene between insect and vertebrate lineages. Phylogenetic analysis indicated that amphioxus akirin represented a true ortholog of the two characterized vertebrate akirin paralogs. Amphioxus akirin, coding 219 amino acids with two nuclear localization signal (NLS) sequences and one 14-3-3 binding motif, was widely expressed in various tissues and up-regulated in response to Escherichia coli (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium) challenges. Furthermore, amphioxus akirin was strictly localized to the nucleus of HEK293T cells in a confocal analysis. Our work identified and characterized for the first time an amphioxus akirin homolog and will promote a better understanding of the evolution and transcriptional network of the akirin gene family.

The D5 region of the intelectin domain is a new type of carbohydrate recognition domain in the intelectin gene family

ABSTRACT Intelectin is a recently characterized soluble galactofuranose‐binding lectin that exists in species ranging from amphioxus to human. Interestingly, intelectin does not contain a canonical carbohydrate‐recognition domain (CRD). Therefore, we designed serial deletions of intelectin in the Chinese amphioxus (Branchiostoma belcheri tsingtauense, AmphiITLN71469) in order to identify functional regions required for carbohydrate binding. Our results revealed that Domain 5 (aa 203–302) was able to bind lipopolysaccarides (LPS) or peptidoglycan (PGN) and agglutinate bacteria as efficiently as the full‐length protein. Three dimensional (3D) atomic models of Domain 5 were generated by ab initio based program QUARK and by Iterative Threading Assembly Refinement (I‐TASSER) programs, in which four amino acids mediating calcium‐binding (G54‐G55‐G56‐E91) were identified by hemagglutination assay. Furthermore, a striking functional conservation of Domain 5 was detected in zebrafish intelectin 1. Taken together, our findings identified for the first time a new CRD domain in intelectin, thereby providing new knowledge leading to a better understanding of pathogen‐host interactions. HighlightsDomain 5 (aa 203–302) was able to bind LPS or peptidoglycan (PGN) and agglutinate bacteria as efficiently as the full‐length protein.Four amino acids mediating calcium‐binding (G54‐G55‐G56‐E91) were identified by hemagglutination assay.

Two distinct phenotypes in pigmented cells of different embryonic origins in eyes of pale ear mice.

The eye has pigmented cells of two different embryonic origins and therefore it is a good model for studying melanosome biogenesis and melanin production/deposition. Pale ear mice bear a mutation in the Hermansky-Pudlak syndrome type 1 (HPS-1) gene and exhibit abnormal eye pigmentation. Here, we reported the delayed and reduced pigmentation in eyes of pale ear mice in early postnatal stages and adulthood. Tyrosinase assay and L-3,4-dihydroxyphenylalanine (L-DOPA) gel staining assay revealed that tyrosinase activity in eyes of pale ear mutants was greatly reduced in early postnatal stages and increased gradually after postnatal day 7 (P7). Further histological examination revealed that hypopigmentation in the retinal pigment epithelium (RPE) and pigment epithelium of the iris and ciliary body, which are derived from the optic cup, was more severe than that in neural crest-derived tissues. In addition, macromelanosomes were exclusively present in neural crest-derived melanocytes of pale ear adults, but absent at early postnatal stages. Taken together, the mutation in the HPS-1 gene could cause two distinct phenotypes in pigmented cells of different embryonic origins. Besides, an increased accumulation of lipofuscin in RPE was also observed.

The Ap3b1 gene regulates the ocular melanosome biogenesis and tyrosinase distribution differently from the Hps1 gene.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder in humans and mice. The pearl (pe) mouse, a mouse model for the human HPS-2, bears a mutation in Ap3b1 gene. Here we investigated the pigmentation in eyes of pearl (pe) mice, and compared it with our previously published data in pale ear (ep) mice. We revealed that the hypopigmentation in eyes of pearl mice was more severe than pale ear mice, especially in the neural crest-derived tissues. However, the total tyrosinase activity in eyes of pearl mice was stronger than pale ear mice, suggesting that the degradation of aberrantly transported tyrosinase in eyes of pearl mice was weaker than that of pale ear mice. Furthermore, the pigmentation in eyes of mice doubly heterozygous for Hps1 and Ap3b1 genes was similar to the wild-type, while the hypopigmentation in iris of double mutant mice was more severe than either single mutant. Besides, we found several previously reported characters in pale ear mice, including macromelanosomes in the neural crest-derived melanocytes and increased accumulation of lipofuscin in the RPE, were absent in pearl mice. Our study indicates that Ap3b1 gene play distinct roles in melanin production and tyrosinase distribution compared with Hps1 gene.

Identification and characterization of an amphioxus matrix metalloproteinase homolog BbMMPL2 responding to bacteria challenge.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases mainly involved in extracellular matrix (ECM) degradation. We have cloned and identified BbMMPL2 as homolog of MMPs from adult amphioxus. Recombinant BbMMPL2 proteins underwent self-processing during refolding in vitro. The final ~23 kDa polypeptide displayed proteolytic activity against ECM components like casein, gelatin, collagen IV and fibrinogen, but not laminin, fibronectin or α1-PI. This activity could be inhibited by GM6001 and TIMP-1/2. In addition, real-time RT-PCR analysis revealed that BbMMPL2 expressed in all issues/organs in adult amphioxus we tested. Its transcription was significantly up-regulated 12 h post immune challenge by Escherichia coli in epidermis and hepatic diverticulum but only slightly increased by Staphyloccocus aureus in epidermis. Furthermore, recombinant BbMMPL2-EGFP expressed in 293T and NIH/3T3 cells showed aggregation in cytoplasm and induced cell death. Our results provided new evidence that MMP was involved in immune response which could be conserved through evolution.

Zebrafish intelectin 1 (zITLN1) plays a role in the innate immune response

Abstract Intelectin displays carbohydrate binding capacity and has been demonstrated to agglutinate bacteria, suggesting its role in innate immunity. It has also been linked to many pathogenic conditions in human. After reporting two amphioxus orthologs and the zebrafish intelectin 2 (zITLN2), here we cloned and characterized zebrafish intelectin 1 (zITLN1). Like zITLN2, zITLN1 also contains a conserved fibrinogen‐related domain (FReD) and a unique intelectin domain (ITLN‐D), expresses in all the tissues tested, with the highest level in intestine, and responds to bacterial challenge in acute phase. We also expressed zITLN1 in E. coli system, and purified recombinant zITLN1 could agglutinate both Gram‐positive and Gram‐negative bacteria in a calcium dependent manner. Its ability to agglutinate Gram‐positive bacteria is stronger than that to Gram‐negative bacteria whereas zITLN2 did not show such preference. This is probably due to the fact that recombinant zITLN1 could bind peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of zITLN1 provided new insight into the evolution and function of the intelectin family. HighlightsAn zebrafish intelectin ortholog, zITLN1 with a fibrinogen‐related domain and a unique intelectin domain was characterized.The zITLN1 is widely expressed with highest level in intestine and upregulated after challenge of Staphylococcus aureus and E.coli.Recombinant zITLN1 binds both LPS and PGN and agglutinates both Gram positive and negative bacteria.

Phylogenetic analysis and expression pattern of the AmphiCaBP-like gene from amphioxus, encoding a novel member of the calmodulin-like subfamily

We characterized a novel gene, AmphiCaBP-like, encoding a putative Ca2+-binding protein (CaBP) with three EF-hand motifs from amphioxus (Branchiostoma belcheri tsingtauense). It exhibits significant similarities with the calmodulin and troponin C proteins from other species. Results of phylogenetic analysis indicated that amphioxus AmphiCaBP-like falls on the base of the troponin C clade. It may be a novel member of the calmodulin-like subfamily. In situ hybridization and RT-PCR analysis showed that AmphiCaBP-like is expressed throughout early development except the stages around the blastula. From neurula stage onward, transcripts of the AmphiCaBP-like are detected in the neural plate, the neural tube, the differentiating somites and the splanchnopleure, as well as the epithelium of the pharynx and gut. It is also expressed in the presumptive, but not the well-developed notochord. In adult amphioxus, the transcripts are found in the epithelial cells of the gut and midgut diverticulus, the wall of coelom, the branchia, the neural cord and gonads.