Cross-reactive HIV-1-neutralizing human monoclonal antibodies identified from a patient with 2F5-like antibodies

The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140(JR-FL). Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW(664-666) core of the 2F5 epitope and two additional upstream residues (L(660,663)). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5--they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.     

The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating protein

The orphan receptor C5L2 has recently been described as a high affinity binding protein for complement fragments C5a and C3a that, unlike the previously described C5a receptor (CD88), couples only weakly to G(i)-like G proteins (Cain, S. A., and Monk, P. N. (2002) J. Biol. Chem. 277, 7165-7169). Here we demonstrate that C5L2 binds the metabolites of C4a and C3a, C4a des-Arg(77), and C3a des-Arg(77) (also known as the acylation-stimulating protein or ASP) at a site distinct from the C5a binding site. The binding of these metabolites to C5L2 does not stimulate the degranulation of transfected rat basophilic leukemia cells either through endogenous rat G proteins or when co-transfected with human G(alpha 16). C3a des-Arg(77)/ASP and C3a can potently stimulate triglyceride synthesis in human skin fibroblasts and 3T3-L1 preadipocytes. Here we show that both cell types and human adipose tissue express C5L2 mRNA and that the human fibroblasts express C5L2 protein at the cell surface. This is the first demonstration of the expression of C5L2 in cells that bind and respond to C3a des-Arg(77)/ASP and C3a. Thus C5L2, a promiscuous complement fragment-binding protein with a high affinity site that binds C3a des-Arg(77)/ASP, may mediate the acylation-stimulating properties of this peptide.     

In vivo 2H/13C flux analysis in metabolism research

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The 5' UTR of protein kinase C epsilon confers translational regulation in vitro and in vivo

We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells.     

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.     

C5L2 is a functional receptor for acylation-stimulating protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.     

Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ⁷-reductase, Δ^8,7-isomerase, and Δ¹⁴-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ²⁴-reductase and Δ^8,7-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10

Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.     

APOA5 and triglyceride metabolism, lesson from human APOA5 deficiency

PURPOSE OF REVIEW: In this review we compare the phenotype and lipoprotein abnormalities of some patients who were found to carry mutations in the APOA5 gene predicted to result in apolipoprotein A-V deficiency. RECENT FINDINGS: The sequencing of the APOA5 gene in patients with primary hypertriglyceridemia, in whom mutations of the LPL and APOC2 genes had been excluded, led to the identification of four families with two different mutations in this gene predicted to result in truncated apolipoprotein A-V. The first mutation (Q148X) was found in a homozygous state in a child with severe type V hyperlipidemia, some clinical manifestations of chylomicronemia syndrome and a slight reduction in plasma postheparin lipoprotein lipase activity. Carriers of a different mutation (Q139X) were recently reported. Four Q139X heterozygotes had type V hyperlipidemia and markedly reduced plasma postheparin lipoprotein lipase activity. The hypertriglyceridemic Q139X heterozygote had other factors that could have contributed to hypertriglyceridemia. ApoB-100 kinetic studies in hypertriglyceridemic Q139X heterozygotes revealed an impairment of very low-density lipoprotein catabolism. SUMMARY: Mutations in the APOA5 gene, leading to truncated apolipoprotein A-V devoid of lipid-binding domains located in the carboxy-terminal end of the protein, if present in the homozygous state, are expected to cause severe type V hyperlipidemia in patients with no mutations in LPL or APOC2 genes. If present in the heterozygous state, these mutations predispose to hypertriglyceridemia in combination with other genetic factors or pathological conditions.     

Activation of annexin 7 by protein kinase C in vitro and in vivo

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.     

Select HIV protease inhibitors alter bone and fat metabolism ex vivo

Human immunodeficiency virus (HIV) therapies have been associated with alterations in fat metabolism and bone mineral density. This study examined the effects of HIV protease inhibitors (PIs) on bone resorption, bone formation, and adipocyte differentiation using ex vivo cultured osteoclasts, osteoblasts, and adipocytes, respectively. Osteoclast activity, measured using a rat neonatal calvaria assay, increased in the presence of nelfinavir (NFV; 47.2%, p = 0.001), indinavir (34.6%, p = 0.001), saquinavir (24.3%, p = 0.001), or ritonavir (18%, p < 0.01). In contrast, lopinavir (LPV) and amprenavir did not increase osteoclast activity. In human mesenchymal stem cells (hMSCs), the PIs LPV and NFV decreased osteoblast alkaline phosphatase enzyme activity and gene expression significantly (p < 0.05). LPV and NFV diminished calcium deposition and osteoprotegrin expression (p < 0.05), whereas the other PIs investigated did not. Adipogenesis of hMSCs was strongly inhibited by saquinavir and NFV (>50%, p < 0.001) and moderately inhibited by ritonavir and LPV (>40%, p < 0.01). Expression of diacylglycerol transferase, a marker of adipocyte differentiation, decreased in hMSCs treated with NFV. Amprenavir and indinavir did not affect adipogenesis or lipolysis. These results suggest that bone and fat formation in hMSCs of bone marrow may be coordinately down-regulated by some but not all PIs.     

Acylation-stimulating protein (ASP)/complement C3adesArg deficiency results in increased energy expenditure in mice

Acylation-stimulating protein (ASP) acts as a paracrine signal to increase triglyceride synthesis in adipocytes. In mice, C3 (the precursor to ASP) knock-out (KO) results in ASP deficiency and leads to reduced body fat and leptin levels yet they are hyperphagic. In the present study, we investigated the mechanism for this energy repartitioning. Compared with wild-type (WT) mice, male and female C3(-/-) ASP-deficient mice had elevated oxygen consumption (VO2) in both the active (dark) and resting (light) phases of the diurnal cycle: +8.9% males (p < 0.05) +9.4% females (p < 0.05). Increased physical activity (movement) was observed during the dark phase in female but not in male KO animals. Female WT mice moved 16.9 +/- 2.4 m whereas KO mice moved 30.1 +/- 5.4 m, over 12 h, +78.4%, p < 0.05). In contrast, there was no difference in physical activity in male mice, but a repartitioning of dietary fat following intragastric fat administration was noted. This was reflected by increased fatty acid oxidation in liver and muscle in KO mice, with increased UCP2 (inguinal fat) and UCP3 (muscle) mRNA expression (p = 0.005 and 0.036, respectively). Fatty acid uptake into brown adipose tissue (BAT) and white adipose tissue (WAT) was reduced as reflected by a decrease in the fatty acid incorporation into lipids (BAT -68%, WAT -29%. The decrease of FA incorporation was normalized by intraperitoneal administration of ASP at the time of oral fat administration. These results suggest that ASP deficiency results in energy repartitioning through different mechanisms in male and female mice.     

Embryonal carcinoma-derived growth factor activates protein kinase C in vivo and in vitro.

We have recently reported that a polypeptide mitogen, the embryonal carcinoma-derived growth factor (ECDGF), induces phosphorylation of the epidermal growth factor (EGF) receptor in intact C3H 10T 1/2 mouse fibroblasts with concomittant loss of high affinity EGF binding sites. This phenomenon appears to be mediated through an activation of protein kinase C. Several groups have described an acidic 80,000 dalton protein substrate of protein kinase C. In this paper, we demonstrate that the addition of ECDGF or the phorbol ester TPA to intact C3H 10T 1/2 cells results in the enhanced phosphorylation of this 80 kd protein in vivo. Furthermore, this response is demonstrable in vitro. Thus the addition of ECDGF, the phorbol ester TPA, protein kinase C or phosphoinositidase C to crude membranes prepared from C3H 10T 1/2 cells resulted in the enhanced phosphorylation of this protein. Data obtained by phosphopeptide mapping of the 80 kd protein show that the ECDGF-induced activation of protein kinase C in our membrane preparations is comparable with that obtained in vivo. The availability of an in vitro system in which this response is preserved should now allow a detailed biochemical analysis of the steps between binding of a mitogen to its receptor and the activation of protein kinase C.