Mannose-binding protein in HIV-seropositive patients does not contribute to disease progression or bacterial infections.

This study set out to investigate whether plasma mannose-binding protein (MBP) deficiency caused by mutations in the MBP gene associates with pyogenic or opportunistic infections in HIV-infected patients. Plasma samples were selected randomly from 131 HIV-infected patients followed prospectively for a period not exceeding 12 months or until death. Plasma MBP concentrations were measured by an ELISA and genotyping was determined by amplification of exon 1 of the MBP gene by polymerase chain reaction (PCR) technology, followed by restriction enzyme analysis and Southern blotting using sequence-specific oligonucleotide probes. Neither MBP concentration nor genotype was found to associate with disease progression or opportunistic infection rate. There was an unexpected increased bacterial infection rate in patients with MBP levels greater than 100 ng/ml and wild type genotype. Thus, MBP does not appear to play a role in HIV infection. MBP is an acute phase reactant and this may explain the higher levels in those with more frequent pyogenic infections.

N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with changes in antibody recognition of the V1/V2 region of gp120.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although NB-DNJ appears to inhibit HIV entry at the level of post-CD4 binding (P.B. Fischer, M. Collin, G.B. Karlsson, W. James, T.D. Butters, S.J. Davis, S. Gordon, R.A. Dwek, and F.M. Platt, J. Virol. 69:5791-5797, 1995), the exact mechanism of action remains to be established. In this study we have examined the effect of NB-DNJ on the structure of recombinant gp120 (rgpl20), expressed in CHO cells, by using a panel of 40 monoclonal antibodies. The levels of binding of antibodies to rgp120 produced in the presence [rgpl20(+)] and absence [rgpl20(-)] of NB-DNJ were compared by enzyme-linked immunosorbent assay and surface plasmon resonance (BIAcore; Pharmacia). The results showed an increase in the binding to rgp120(+) of antibodies directed against the C1 and C2 regions and a decrease in the binding of antibodies directed against the V1/V2 loops compared with antibody binding to rgpl20(-). A decrease in the binding to rgpl20(+) of antibodies directed against discontinuous epitopes was also observed. No differences were seen in the binding of antibodies directed against the crown of the V3 loop and the C4 region of gp120. Treatment of rgpl20 with alpha-glucosidases I and II had no effect on the differential binding observed, whereas treatment with sialidase abolished the differences seen in the binding of antibodies directed against the C1 and C2 regions of gp120. In addition to these findings, rgpl20(+) showed increased sensitivity to proteases released by CHO cells during expression, as well as to exogenous thrombin. Taken together, the data presented in this paper suggest that production of gp120 in the presence of NB-DNJ affects the conformation of the Vl/V2 loops of gpl20, as well as the overall charge of the C1 and C2 regions. These effects may play a role in the previously described NB-DNJ-mediated inhibition of HIV entry at the level of post-CD4 binding.

N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.

Affinity and kinetic analysis of the bovine plasma C-type lectin collectin-43 (CL-43) interacting with mannan.

Collectins are C-type lectins which have been implied to play an important role in the innate immune defence against microorganisms. The critical discriminatory event in the opsonization of microorganisms by collectins is the interaction of the C-type lectin domain with microbial carbohydrates. Surface plasmon resonance measurements allow for quantitative real-time measurements of binding interaction between immobilized carbohydrate and unlabelled lectin in solution. Binding analysis were carried out with purified collectin-43 (CL-43) which structurally is the simplest collectin consisting of only three polypeptides each terminating in a C-type lectin domain. The target was immobilized yeast mannan. The molecular mass of native CL-43 was determinated by mass spectroscopy to 99.8 kDa. The dissociation rate (kdiss) of the C-type lectin-carbohydrate binding was fast (1.19-1.36 x 10(-2) second-1), and the association rate (kass) was 4.37-5.07 x 10(5) M-1 second-1. The equilibrium constant for dissociation (Kd) was 2.68-2.72 x 10(-8) M.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.

Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.

The glycosylation of the circulating immunoglobulin-gamma (IgG) antibody molecules changes in rheumatoid arthritis. The extent of the changes correlates with the disease severity and reverses in remission. We demonstrate here that the alteration in glycosylation associated with rheumatoid arthritis can create a new mode for the interaction of IgG with complement through binding to the collagenous lectin mannose-binding protein (MBP). Rheumatoid arthritis is associated with a marked increases in IgG glycoforms that lack galactose (referred to as G0 glycoforms) in the Fc region of the molecule and that terminate in N-acetyl glucosamine (GlcNAc). We show, using nuclear magnetic resonance (NMR) and X-ray data, that these terminal GlcNAc residues become accessible for MBP binding. We further demonstrate that multiple presentation of IgG-G0 glycoforms to MBP results in activation of the complement. This suggests that a contribution to the chronic inflammation of the synovial membrane could arise from the localization of the IgG-G0 glycoforms in the affected joint and from resulting activation of complement.

Mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice.

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type 1 (HSV-1) in vitro. We decided to investigate the role of mammalian lectins in infection with herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein, MBP), belonging to the collectin family of lectins, were examined. Four week-old BALB/c mice were injected subcutaneously with 100 micrograms bovine conglutinin or 50 micrograms human MBP 1 day before intravenous infection with 5 x 10(4) PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP, whereas no effect was seen on days 1 and 5. In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations of the collectins. The concentrations used were similar to those used by us in vivo, and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins. In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitable binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions with an alternative port of entry into cells leading to infection enhancement.

Complement activation upon binding of mannan-binding protein to HIV envelope glycoproteins.

OBJECTIVE: Retroviruses can activate the complement system in the absence of antibodies, and the purpose of this study was to examine whether the serum collection, mannan-binding protein (MBP), could mediate such complement activation. DESIGN: Virus envelope proteins gp120 and gp110 from HIV-1 and HIV-2 were incubated in microtitre wells coated with anti-gp120 or anti-gp110 antibodies. After further incubation with serum, complement activation was measured as deposition of complement factor C4 and C3 onto the wells. Deposited C4 and C3 were detected with enzyme-labelled antibodies. Normal human serum depleted of endogenous lectins by affinity chromatography was used as the complement source. Serum from C1q-deficient patients was used in some experiments. Complement activation was then assessed with and without prior addition of MBP to the wells. Complement activation was also correlated with the quantity of endogenous MBP in a number of normal sera. RESULTS: Complement activation by HIV envelope glycoproteins was found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins produced in insect cells. Non-glycosylated recombinant envelope proteins produced in Escherichia coli did not induce this type of complement activation. CONCLUSIONS: Activation of the classical complement pathway by retrovirus envelope proteins can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins.