Epitope mapping and cross-reactivity analysis of the monoclonal antibodies against hexon protein of human adenovirus type 3.

Human adenovirus 3 (HAdV-3) occurs epidemically in China in recent years. It can cause life-threatening infection in young children and immunocompromised patients. Development of reliable diagnostic reagents for adenovirus infection is important in surveillance and control of the infection. In this study, three monoclonal antibodies (MAbs) 5D4, 7B2 and 3D10 were generated against the recombinant HAdV-3 hexon protein. Western-blots analysis showed that all three MAbs recognized the native HAdV-3 hexon protein, but only 5D4 reacted with the native HAdV-3 virus particles by indirect enzyme-linked immunosorbent assay (ELISA). The epitopes recognized by these MAbs were further investigated by epitope analysis with a series of peptides of the hexon protein. The epitope for 3D10 was located to the sequence (339)GVLAGQASQLNA(350) and present in all the currently recognized serotypes of human adenovirus. The MAb 7B2 showed broad reactivity with the peptides from group B, group E, and most serotypes of group D, but not with those from group A, C and F adenoviruses. The epitope of 5D4, located in the hypervariable region 3 (HVR3), was only shared by HAdV-3 and HAdV-7. Further ELISA demonstrated that 5D4 only recognized HAdV-3 and HAdV-7 virus particles. The MAbs and the identified epitopes may play roles in clinical serotype-specific diagnosis, viral structure analysis, as well as in the identification of new serotypes of human adenovirus.

A novel C1q family member of amphioxus was revealed to have a partial function of vertebrate C1q molecule.

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and adaptive immunities. Its globular signature domain is also found in a variety of noncomplement protein that can be grouped together as a C1q family. In this study, we have cloned and identified a novel C1q family member in cephalochordate amphioxus and named it as AmphiC1q1. The high transcriptional levels of this gene were detected during all stages of embryonic development, and the section in situ hybridization demonstrated that AmphiC1q1 was mainly expressed in the ovary, intestine, and nerve system of mature individuals. The transcript of AmphiC1q1 was up-regulated by LPS and gram-negative bacteria, but hardly by lipoteichoic acid and Staphylococcus aureus. The recombinant AmphiC1q1 protein could not bind with N-acetyl-glucosamine and did not possess hemagglutinating activity, indicating that AmphiC1q1 could not act as its lamprey homologue. But both the full-length protein and its truncated globular domain of C1q protein could interact with LPS. Moreover, recombinant AmphiC1q1 protein could inhibit collagen-induced platelet aggregation, but the truncated globular C1q domain protein would not, indicating that the blocking activity of AmphiC1q1 protein was via the collagen region of the protein. Our study on the primitive form of C1q family in protochordate will shed a light on understanding the gradual functional evolution of C1q family and eventual formation of mammalian homologues.

A short-form C-type lectin from amphioxus acts as a direct microbial killing protein via interaction with peptidoglycan and glucan.

To investigate the evolution and immune function of C-type lectin in amphioxus, the primitive representative of the chordate phylum, we identified three C-type lectins consisting solely of a carbohydrate recognition domain and N-terminal signal peptide and found that they had distinct express patterns in special tissues and immune response to stimulations analyzed by quantitative real-time PCR. We characterized the biochemical and biological properties of AmphiCTL1, which was dramatically up-regulated in amphioxus challenged with Staphylococcus aureus, Saccharomyces cerevisiae, and zymosan. Immunohistochemistry demonstrated that the localization of AmphiCTL1 protein was exclusively detected in the inner folding tissues of the hepatic diverticulum. Recombinant AmphiCTL1 was characterized as a typical Ca2+-dependent carbohydrate-binding protein possessing hemagglutinating activity, preferentially bound to all examined four Gram-positive bacteria and two yeast strains, but had little binding activity toward four Gram-negative bacteria we tested. It aggregated S. aureus and S. cerevisiae in a Ca2+-dependent manner and specifically bound to insoluble peptidoglycan and glucan, but not to LPS, lipoteichoic acid, and mannan. Calcium increased the intensity of the interaction between AmphiCTL1 and those components, but was not essential. This lectin directly killed S. aureus and S. cerevisiae in a Ca2+-independent fashion, and its binding to microorganism cell wall polysaccharides such as peptidoglycan and glucan preceded microbial killing activity. These findings suggested that AmphiCTL1 acted as a direct microbial killing C-type lectin through binding microbial targets via interaction with peptidoglycan and glucan. Thus, AmphiCTL1 may be an evolutionarily primitive form of antimicrobial protein involved in lectin-mediated innate immunity.

Molecular and biochemical characterization of galectin from amphioxus: primitive galectin of chordates participated in the infection processes.

A novel F4-carbohydrate recognition domain (CRD)-linker-F3-CRD-type bi-CRD Branchiostoma belcheri tsingtauense galectin (BbtGal)-L together with its alternatively spliced mono-CRD isoform BbtGal-S from amphioxus intestine was encoded by a 9488-bp unique gene with eight exons and seven introns. The recombinant proteins of BbtGal were found to have beta-galactoside-binding activity, indicating that BbtGal was a member of the galectin family. Phylogenetic analysis of this gene along with its splicing form and genome structure suggested that the BbtGal gene was the primitive form of the chordate galectin family. Real-time polymerase chain reaction analyses (PCR) indicated that BbtGal mRNA was expressed during all stages of embryonic development. In terms of tissue distribution, BbtGal-L mRNA was mainly expressed in the immunity-related organs, such as hepatic diverticulum, intestine, and gill, but BbtGal-S was ubiquitously expressed in all tissues. The expression of BbtGal-L mRNA was elevated after acute challenge with various microorganisms, but BbtGal-L only bound to specific bacteria. The immune function of BbtGal was consistent with its localization both outside and inside the cell. Our study on amphioxus galectin may help further understanding of the evolution of chordate galectin in terms of host-pathogen interaction in the immune system.

New insights on macrophage migration inhibitory factor: based on molecular and functional analysis of its homologue of Chinese amphioxus.

Macrophage migration inhibitory factor (MIF) is an intricate cytokine. Many questions about it are not fully resolved. In order to identify the role of MIF in Chinese amphioxus, its genomic organization, transcription pattern and enzymatic activity were studied. It's found that MIF has multi-copy gene number in the Chinese amphioxus genome and special transcription pattern in reproductive organs. Interestingly, the recombinant Bbt-MIF has tantomerase and redox activity, but fails to utilize GSH to reduce insulin instead of DTT, strikingly different from MIF in mammalian. All these results indicate that MIF gene must have undergone important changes in structure and function during the transition of invertebrate/vertebrate and might exert important role in this primitive species, which may be quite different from those found in vertebrate.