Complement Inhibitors Vitronectin and Clusterin Are Recruited from Human Serum to the Surface of Coronavirus OC43-Infected Lung Cells through Antibody-Dependent Mechanisms

Little is known about the role of complement (C’) in infections with highly prevalent circulating human coronaviruses such as OC43, a group of viruses of major public health concern. Treatment of OC43-infected human lung cells with human serum resulted in C3 deposition on their surfaces and generation of C5a, indicating robust C’ activation.
Real-time cell viability assays showed that in vitro C’-mediated lysis of OC43 infected cells requires C3, C5 and C6 but not C7, and was substantially delayed as compared to rapid C’-mediated killing of parainfluenza virus type 5 (PIV5)-infected cells.
In cells co-infected with OC43 and PIV5, C’-mediated lysis was delayed, similar to OC43 infected cells alone, suggesting that OC43 infection induced dominant inhibitory signals.
When OC43-infected cells were treated with human serum, their cell surfaces contained both Vitronectin (VN) and Clusterin (CLU), two host cell C’ inhibitors that can alter membrane attack complex (MAC) formation and C’-mediated killing.
VN and CLU were not bound to OC43-infected cells after treatment with antibody-depleted serum. Reconstitution experiments with purified IgG and VN showed that human antibodies are both necessary and sufficient for VN recruitment to OC43-infected lung cells-novel findings with implications for CoV pathogenesis.

Additivity in effects of vitronectin and monoclonal antibodies against alpha-helix F of plasminogen activator inhibitor-1 on its reactions with target proteinases.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators.
Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5.
Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2.
Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases.
The same step in the reaction mechanism remains to limit for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki).
These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.

Activated vitronectin as a target for anticancer therapy with human antibodies.

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites.
Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs).
In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin.
One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues.
Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.

New insights into heparin binding to vitronectin: studies with monoclonal antibodies.

Vitronectin is a plasma glycoprotein that binds to a variety of ligands. There is considerable debate regarding the dependency of these binding interactions upon the conformational status of vitronectin, the role of multimerization and how the binding of different ligands can change vitronectin’s conformational state.
We have developed a method of capturing vitronectin directly from fresh plasma using solid-phase monoclonal antibodies. Various biotin-labelled secondary monoclonal antibodies were used to quantify the bound vitronectin and to measure its degree of denaturation.
Using these tools we demonstrated that one monoclonal antibody partially denatured vitronectin without direct multimerization.
Treatment of vitronectin in plasma with soluble heparin produced a similar degree of denaturation. These results led to a proposed adaptation of the unfolding/refolding pathways for chemically denatured vitronectin originally presented by Zhuang and co-workers in 1996 [Zhuang, Blackburn and Peterson (1996) J. Biol. Chem. 271, 14323-14332 and Zhuang, Li, Williams, Wagner, Seiffert and Peterson (1996) J. Biol. Chem. 271, 14333-14343]. The adapted version allows for the production of a more stable partially unfolded intermediate, resulting from the binding of particular ligands.
We also demonstrated that the avidity of heparin binding to vitronectin is governed by both the conformational state of the monomer and multimerization of the molecule.

Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding.

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F.
In order to elucidate this mechanism,
we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1).
One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1.
When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1.

Vitronectin antibody

70R-50548 Fitzgerald 100 ul 244 EUR

Vitronectin antibody

20R-1396 Fitzgerald 10 mg 275 EUR

Vitronectin antibody

70R-10610 Fitzgerald 500 ug 492 EUR

Vitronectin antibody

70R-14336 Fitzgerald 100 ug 322 EUR

Vitronectin antibody

10-1962 Fitzgerald 200 ul 543 EUR

Vitronectin antibody

10-1963 Fitzgerald 200 ul 543 EUR

Vitronectin antibody

10-1964 Fitzgerald 200 ul 769 EUR

Vitronectin antibody

10-1965 Fitzgerald 200 ul 543 EUR

Vitronectin antibody

10R-8488 Fitzgerald 100 ul 393 EUR

Vitronectin Antibody

48876-100ul SAB 100ul 333 EUR

Vitronectin Antibody

48876-50ul SAB 50ul 239 EUR

Vitronectin Antibody

abx023996-200ug Abbexa 200 ug 578 EUR

Vitronectin Conjugated Antibody

C48876 SAB 100ul 397 EUR

anti- Vitronectin antibody

FNab09415 FN Test 100µg 548.75 EUR

Vitronectin antibody (HRP)

60R-1036 Fitzgerald 100 ug 358 EUR

Anti-Vitronectin antibody

PAab09415 Lifescience Market 100 ug 386 EUR

Vitronectin (VTN) Antibody

20-abx327315 Abbexa
  • 314.00 EUR
  • 244.00 EUR
  • 100 ug
  • 50 ug

Vitronectin (VTN) Antibody

20-abx131678 Abbexa
  • 425.00 EUR
  • 133.00 EUR
  • 1205.00 EUR
  • 578.00 EUR
  • 328.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Vitronectin (VTN) Antibody

20-abx130338 Abbexa
  • 439.00 EUR
  • 133.00 EUR
  • 1247.00 EUR
  • 592.00 EUR
  • 328.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Vitronectin (VTN) Antibody

20-abx125407 Abbexa
  • 495.00 EUR
  • 704.00 EUR
  • 356.00 EUR
  • 100 ul
  • 200 ul
  • 50 ul

Vitronectin (VTN) Antibody

20-abx127082 Abbexa
  • 411.00 EUR
  • 592.00 EUR
  • 100 ul
  • 200 ul
Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators.
These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.