Thermodynamic signatures of the antigen binding site of mAb 447-52D targeting the third variable region of HIV-1 gp120.

The third variable region (V3) of HIV-1 gp120 plays a key role in viral entry into host cells; thus, it is a potential target for vaccine design. Human monoclonal antibody (mAb) 447-52D is one of the most broadly and potently neutralizing anti-V3 mAbs. We further characterized the 447-52D epitope by determining a high-resolution crystal structure of the Fab fragment in complex with a cyclic V3 and interrogated the antigen-antibody interaction by a combination of site-specific mutagenesis, isothermal titration calorimetry (ITC) and neutralization assays. We found that 447-52D's neutralization capability is correlated with its binding affinity and at 25 °C the Gibbs free binding energy is composed of a large enthalpic component and a small favorable entropic component. The large enthalpic contribution is due to (i) an extensive hydrogen bond network, (ii) a π-cation sandwiching the V3 crown apex residue Arg(315), and (iii) a salt bridge between the 447-52D heavy chain residue Asp(H95) and Arg(315). Arg(315) is often harbored by clade B viruses; thus, our data explained why 447-52D preferentially neutralizes clade B viruses. Interrogation of the thermodynamic signatures of residues at the antigen binding interface gives key insights into their contributions in the antigen-antibody interaction.

Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gp120 helper epitopes.

Glycosylation of HIV-1 envelope gp120 determines not only the proper structure, but also the immune responses against this Ag. Although glycans may be part of specific epitopes or shield other epitopes from T cells and Abs, this study provides evidence for a different immunomodulatory function of glycans associated with gp120 residues N230 and N448. These glycans are required for efficient MHC class II-restricted presentation of nearby CD4 T cell epitopes, even though they are not part of the epitopes. The glycans do not affect CD4 T cell recognition of more distant epitopes and are not essential for the proper folding and function of gp120. Data on CD4 T cell recognition of N448 mutants combined with proteolysis analyses and surface electrostatic potential calculation around residue N448 support the notion that N448 glycan near the epitope's C terminus renders the site to be surface accessible and allows its efficient processing. In contrast, the N230 glycan contributes to the nearby epitope presentation at a step other than the proteolytic processing of the epitope. Hence, N-glycans can determine CD4 T cell recognition of nearby gp120 epitopes by regulating the different steps in the MHC class II processing and presentation pathway after APCs acquire the intact gp120 Ag exogenously. Modifications of amino acids bearing glycans at the C termini of gp120 helper epitopes may prove to be a useful strategy for enhancing the immunogenicity of HIV-1 envelope gp120.

An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene.

An expression release system for heterogeneous proteins in Escherichia coli mediated by the colicin release gene(kil gene) was constructed. The system is based on the ability of the Kil protein to release periplasmic proteins into the growth medium. beta-lactamase, an E.coli periplasmic protein, and prolyl endopeptidase (PEP), a periplasmic protein of Aeromonas punctata subsp. punctata were used as report proteins. Commonly, these two proteins are seldom released into the growth medium. The results indicated that the released amounts of the beta-lactamase and of the prolyl endopeptidase were nearly 5 fold and 4 fold than control, respectively.

The Thioredoxin Reductase-deficient E.coli Mutant Enhances Expression into Solution of Recombinant Proteins Containing Cys Residues.

A 3D artificial protein, a salmon calcitonin hexa-polymer, a salmon calcitonin octo-polymer and a human prourokinase, was expressed in the cytoplasma of E.coli GJ980(trxB(-)) mutant. These recombinant proteins containedcysteine residues of different length ranging from 12-22 residues. The mutation was mapped to the gene for thioredoxin reductase(trxB) and was found to eliminate the activity of this enzyme, which was thought to contribute to the sulfhydryl reducing potential of the cytoplasm. Recombinant salmon calcitonin hexapolymer, salmon calcitonin octo-polymer and human prourokinase had more soluble form in cytoplasm of GJ980 mutants than in wild-type strain, while 3D-protein, which has nocysteine residue, still remain in insoluble form. Results indicate the GJ980(trxB(-)) strain allowed the formation of disulphide bonds in the cell cytoplasm which is believed to encourage correct folding and soluble expression of the recombinant proteins.

Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

Interaction of C17orf25 with ADP-ribose pyrophosphatase NUDT9 detected via yeast two-hybrid method.

The gene C17orf25 was isolated from the liver by RACE PCR. nudt9 gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA library. NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose as its substrate. Fusion expression of C17orf25 and GFP and computer analysis showed that C17orf25 was probably located in mitochondria. Furthermore, C17orf25 may suppress the cell growth by interaction with NUDT9.

Construction of a Novel Engineering Strain E.coli G830,which is Adoptable to High-cell-density Fermentation.

A novel engineered strain G830 adoptable to high-cell-density fermentation by integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin gene) into thr operon in the chromosome of PA1 blocking Pta-Ack metabolic pathway through the homologous recombination between the homologous fragments of integrated plasmid and that of chromosome. The engineered strain G830 was characterized by phenotype observation, PCR, thr mutant, acetate acid detection, Western blotting and VHb activity assays. In high dentity fermentation, the cellular respiration, energy metabolism, highest bacterial density and dry bacteria weight of the G830 strain were markedly better than control strains PA1 and BL21. The expression of recombinant prolyl endopeptidase (PEP) in G830 and PA1 under the above condition was high and stable. Their growth situation and fermentation parameters were similar with their parental strains without plasmids, and resided plasmids maintained stably in those strains. It revealed that the integral VHb and acetate metabolism pathway (Pta-Ack) block improved the growth of host strain under low-dissolved-oxygen conditions, enhanced the recombinant proteins production and reduced the accumulation of acetate harmful to bacterial growth. In conclusion, the novel engineering strain G830 was adoptable to high cell density fermentation.

[Mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ agonists on transforming growth factor ß1 in adult skin fibroblasts].

OBJECTIVE: To study the mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on transforming growth factor ß1 (TGF-ß(1))-induced scarring of skin. METHODS: Fibroblasts isolated from healthy adult skin were cultured in vitro and divided into blank control group (serum-free DMEM culture), TGF-ß(1) group (with stimulation of 10 ng/mL TGF-ß(1) for 48 hours), troglitazone group (with the same treatment as in TGF-ß(1) group after stimulation of 10 µmol/L troglitazone for 2 hours), and 15-dioxygen prostaglandin J2 (15d-PGJ2) group (with the same treatment as in TGF-ß(1) group after stimulation of 10 µmol/L 15d-PGJ2 for 2 hours) according to the stimulation added into DMEM. The expression of connective tissue growth factor (CTGF) was determined with Western blot. The mRNA levels of CTGF, matrix metalloproteinase-1 (MMP-1) and platelet-derived growth factor (PDGF) were determined with real-time fluorescence RT-PCR. Data were processed with one-way analysis of variance. RESULTS: The expression of CTGF at mRNA and protein levels in skin fibroblasts were significantly increased in TGF-ß(1) group as compared with control group; while expression of CTGF at mRNA and protein levels in 15d-PGJ2 and troglitazone groups were significantly decreased as compared with that in TGF-ß(1) group. The mRNA level of MMP-1 in TGF-ß(1) group (0.193 ± 0.051) was obviously lower than that in blank control group (1.281 ± 0.195, F = 12.811, P < 0.01), while the mRNA levels of MMP-1 in troglitazone group (0.417 ± 0.043) and 15d-PGJ2 group (0.485 ± 0.027) were significantly increased as compared with that in TGF-ß(1) group (F = 12.811, P values all below 0.01). The mRNA level of PDGF in TGF-ß(1) group (1.044 ± 0.237) was obviously higher than that in control group (0.349 ± 0.057, F = 16.848, P < 0.01), while the levels in troglitazone group (0.677 ± 0.055) and 15d-PGJ2 group (0.511 ± 0.017) were significantly decreased as compared with that in TGF-ß(1) group (F = 16.848, P values all below 0.01). CONCLUSIONS: The inhibitory effect of activated PPARγ on the expression of CTGF induced by TGF-ß(1) may be the main mechanism of its inhibitory effect on TGF-ß(1)-induced scarring on skin, and its influence on MMP-1 and PDGF may also be one of the underlying mechanisms.

Identification of an N-linked glycosylation in the C4 region of HIV-1 envelope gp120 that is critical for recognition of neighboring CD4 T cell epitopes.

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.

HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.