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Chemical Immunology Research Center, Departments of Pathology and Medicine, University of Texas Medical School, Houston, Texas, USA
1Correspondence: Chemical Immunology Research Center, Department of Pathology, University of Texas Medical School, 6431 Fannin, Houston, TX 77030, USA. E-mail: Sudhir.Paul{at}uth.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Key Words: SLE • neuroimmune regulation • catalytic antibodies
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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. Smooth muscle relaxant action of the neuropeptide vasoactive intestinal polypeptide (VIP) is blunted by a model monoclonal antibody with VIP cleaving activity (2)
. Recent studies indicate that VIP mediates important immunoregulatory effects. For example, the peptide regulates the synthesis of various cytokines by T helper cells and the expression of immunomodulatory molecules such as the Fas ligand on the surface of the cells. (3
4
5)
. Tissue concentrations of VIP are altered in humans and experimental animals with autoimmune disease (6
, 7)
, and induction of VIP receptor dysfunction using transgenic and knockout techniques induces changes in T cell responsiveness to exogenous antigens (8
, 9)
. Direct evidence for the likely importance of VIP in regulating autoreactive immune responses is provided by animal studies showing that exogenous administration of the peptide reduces amelioration of collagen-induced rheumatoid arthritis (10)
.
No information is available about the presence and characteristics of VIPases in classical autoimmune diseases, but other catalytic antibodies have been linked to autoimmune, lymphoproliferative, and transfusion-induced diseases (11
12
13
14
15
16)
. Defective Fas receptor expression has been implicated as the cause for increased esterolytic antibody synthesis in autoimmune mouse strains immunized with phosphonate transition state mimics of ester bond cleavage (17
, 18)
. Among the known autoantigen targets for catalytic antibodies (reviewed in ref 19
), VIP is unique in its ability to regulate innate and adaptive immune responses. Immunoregulatory disturbances induced by VIPases therefore can be conceived to play a role in loss of tolerance to self-antigens. We describe the association of VIPase autoantibodies with human autoimmune disease and Fas ligand defective, lupus-prone gld mice. Despite low VIP binding affinity, the VIPases displayed specific cleavage of this polypeptide, suggesting that specificity may arise from the chemical steps necessary for the catalytic cycle. Recombinant Fv constructs with VIPase activity were isolated and characterized from a lupus phage display library. Enzyme inhibitor and covalent binding studies suggested the serine protease-like character of the VIPases. These observations open the route toward elucidation of the role of VIPase autoantibodies in the pathogenesis of autoimmune disease.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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for clinical characteristics of SLE, ATh, and HIV-1 positive subjects). Adult gender-matched individuals without a history of autoimmune disease served as controls. Sera from young mice (5–8 wk) and old C3H/gld mice (26–28 wk) were prepared from blood obtained from the retro-orbital plexus (the gld mutation in the Fas ligand results in uncontrolled lymphoproliferation due to deficient apoptosis). Control sera were from wild-type C3H/++ mice and BALB/c mice (Jackson Laboratory, Bar Harbor, ME).
Antibodies
IgG was purified from serum by affinity chromatography on protein-G Sepharose (12)
. The characteristics of the phage displayed lupus Fv library have been described (20)
. Selection of phages (2x1014 CFU) was by binding to synthetic VIP immobilized on Affi-gel 10 (Biorad, Hercules, CA, USA; 0.2 mg/ mL settled gel) at 4°C in the presence of 1 mM di-isopropyl fluorophosphate (Sigma, St. Louis, MO, USA) in PBS (10 mM sodium phosphate, 0.137 mM NaCl, 2.7 mM KCl, pH 7.4) containing 2% skim milk (21)
. Following phage binding (14 h, 4°C), the column was washed with PBS until A280 was <0.01. Bound phages were eluted with 0.1 M glycine-HCl, pH 2.7 (2 mL fractions neutralized with 2 M Tris base). Soluble Fv was purified by Ni-NTA chromatography from periplasmic extracts of HB2151 cells harboring the phagemid DNA (20)
. Fv expression levels were 0.11–0.48 mg/L. cDNA was sequenced by the dideoxynucleotide chain termination method and sequences were analyzed for germline gene origin and subgroup assignment using NCBI IgBlast and Kabat databases (http://www.ncbi.nlm.nih.gov/BLAST; http://immuno.bme.nwu.edu). SDS-PAGE of IgG and Fv preparations, silver staining, and immunoblotting with anti-human IgG or anti-myc antibodies as in refs 22
, 23
.
Proteolysis assays
VIP was synthesized by solid-phase FMOC chemistry. Preparation of [tyr10-125I]VIP was as in ref 23
. [tyr10-125I]VIP (30,000 c.p.m, 
50 pM) was incubated with IgG at 37°C in 0.2 mL 50 mM Tris-HCl, 100 mM glycine, 0.025% Tween-20, 0.1% bovine serum albumin (BSA), pH 7.7, for 14–16 h in triplicate and peptide hydrolysis was estimated as the radioactivity soluble in 10% trichloroacetic acid (TCA) corrected for background acid-soluble radioactivity in reaction mixtures containing diluent instead of IgG (6.2 ± 2.5% of total radioactivity, mean ± SD, N=7 assays). Samples showing hydrolysis >7.5% the background acid-soluble radioactivity (corresponding to 3 SD values > background) were designated VIPase positive. Peptide breakdown estimated by this method is essentially identical to that obtained by reversed-phase HPLC (22)
. Confirmatory HPLC was on a Novapak C18 column (Waters, Milford, MA, USA) using a gradient of acetonitrile in 0.1% trifluoroacetic acid. Initial rate data at varying VIP concentration (10 nM–120 µM) were fitted to the Michaelis-Menten-Henri equation by nonlinear regression (Prism, Graphpad). Eledoisin and bombesin used as alternate substrates were from Bachem (Bubendorff, Switzerland). Cleavage of Pro-Phe-Arg-methylcoumarinamide (MCA) substrates (Peptides International, Louisville, KY, USA) was assayed in triplicate by incubation with IgG in 96-well plates (Microfluor W, Dynatech, Vienna, VA, USA) at 37°C in 50 µL 50 mM Tris-HCl, 100 mM glycine, pH 7.7, 0.025% Tween-20 followed by measurement of fluorescence of the leaving group, aminomethylcoumarin (
ex 370 nm, em 460 nm) (12)
. Product concentration was computed from a standard curve constructed using authentic aminomethylcoumarin (20 fluorescence units/µM/50 µL). 125I-Labeled thyroglobulin cleavage was determined by SDS electrophoresis (12)
. For immunoadsorption studies (22)
, human IgG preparations (9.2 µg or 92 µg protein; 230 µL) were treated with goat anti-human IgG (H + L) conjugated to Sepharose 4B (Zymed, San Francisco, CA, USA; 1 mL settled gel, IgG binding capacity 1–2 mg/mL gel) in 50 mM Tris-HCl, 0.1 M glycine, 0.025% Tween-20 (3 h, 4°C). The supernatant was recovered and assayed for VIPase activity. Immunoadsorption of mouse IgG was done similarly (C3H/gld IgG, 20 µg) using 0.2 mL of anti-mouse IgG conjugated to Sepharose 4B (Zymed, 1 mL settled gel, IgG binding capacity 3.3 mg/mL).
Phosphonate diester binding
Synthesis of diphenyl N-[6-(biotinamido)hexanoyl]amino(4-amidinophenyl)methanephosphonate (Bt-Z) and analysis of its covalent binding by IgG and Fv preparations were as in ref 20
. The reaction was allowed to proceed for 60 min; samples were kept at 90°C (2 min), then subjected to SDS electrophoresis, electroblotting, and detection of biotin by staining with streptavidin-peroxidase using a chemiluminescence kit.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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) were analyzed for [tyr10-125I]VIP cleaving activity. The activity was not detected in IgG from wild-type, aged C3H/++ mice (Fig. 1)
. In comparison, VIPase activity was evident in each of the IgG preparations from aged C3H/gld mice (P<0.01 compared with C3H/++ mice; Mann Whitney U test, 2-tailed). No VIPase activity was observed in 6 young C3H/gld mice and 9 aged BALB/c mice. VIPase activities were compared in control humans without autoimmune disease, lupus patients, ATh patients, and subjects expressing an immune response to an infectious agent (HIV-1). No VIPase activity was observed in IgG preparations from control subjects (Fig. 1)
. Eleven of 12 HIV-1-positive subjects were negative for the activity, with the twelfth subject expressing marginal activity. In comparison, VIPase activity was observed in 8/8 ATh patients and 7/10 lupus patients (P<0.01 for both groups vs. control subjects or HIV-1-positive subjects).
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Immunoadsorption using immobilized anti-IgG antibodies was done at two concentrations of lupus IgG code 523, 1.22 µM IgG and a 100-fold more dilute solution, 12.2 nM IgG. This resulted in near-complete removal of the VIPase activity (100.0±2.7% and 98.7±9.3%, respectively). Similarly immunoadsorption of ATh IgG code 584 and pooled C3H/gld IgG resulted in removal of 97.8 ± 2.7% and 91.7 ± 13.1% VIPase activity, respectively. Previous studies of VIPase IgG purified from serum exactly as in the present report provide additional assurance that the VIPase activities are attributable to IgG. The VIPase activity of the IgG is retained in Fab fragments and in reduced and alkylated light chains purified by gel filtration in denaturing conditions, and it is enriched by further affinity chromatography on immobilized VIP (22)
.
Purified Fv clones from the human lupus library were analyzed to determine whether the activity belongs to autoantibody variable domains. In addition to randomly picked Fv clones (n=7), 11 Fv clones were obtained by selecting the phage library on an immobilized VIP column under conditions that minimize peptide cleavage (low temperature, 1 mM DFP). None of seven Fv clones isolated without selection on VIP displayed detectable VIPase activity. Of 11 selected Fv clones, 4 displayed VIPase activity ranging from 15.3 to 53.9% cleavage/nM Fv (P=0.002, Mann-Whitney U test, vs. unselected Fv; Fig. 1
).
Reversed-phase HPLC confirmed that the TCA precipitation method accurately measures fragmentation of VIP (Fig. 2
). HPLC of (tyr10-125I)VIP incubated with a catalytic mouse IgG sample (i.d. 1178) yielded a major and a minor radiolabeled peak corresponding to fragments of [tyr10-125I]VIP (retention times 4.5 min and 11.5 min compared with 13.5 min for intact [tyr10-125I]VIP), amounting to 75.1% of the total recovered radioactivity. This value was close to the extent of [tyr10-125I]VIP cleavage determined by TCA precipitation (75.8%).
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Catalytic properties
Essentially linear increases in the rates of [tyr10-125I]VIP cleavage at increasing IgG and Fv concentrations were observed (data not shown). Study of rates as a function of VIP concentration indicated the reaction to be saturable and consistent with the Michaelis-Menten equation (Fig. 3
; v=(Vmax·[Antigen])/(Km+[Antigen]); Vmax, velocity at saturating antigen concentration; Km Kd; for polyclonal IgG, the observed Km is the average and observed Vmax, the sum of the values for the individual VIPase subpopulations). The apparent Km values were in the high µM range for lupus IgG samples, with somewhat lower values evident for ATh and C3H/gld IgG samples (Table 1
). The kinetic characteristics of the two lupus VIPase Fv clones (DM506 and DM408, GenBank accession numbers AF509586 and AF509587, respectively) were consistent with the method used for their isolation, i.e., selective trapping of high-affinity VIP binders (Table 2
). The Km of these clones was smaller and their Vmax was comparable to the values for lupus IgG. As the Fv constructs are monoclonal, their turnover (kcat) and kinetic efficiencies (kcat/Km) can be computed (Table 2)
; these values compare favorably to other examples of proteolytic antibodies (19)
. Consumption of multiple mol of VIP/mol Fv over the course of the reaction (31 and 18 mol for clones DM408 and DM506, respectively) satisfies a defining requirement for a catalyst, i.e., turnover.
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cDNA sequencing allowed determination of the germline gene counterparts of the Fv variable regions and the extent of somatic diversification. Five and 14 replacement mutations are evident in the VH and VL regions of clones DM408 and DM506, respectively, and the ratio of replacement/silent mutations (R/S) in the CDRs is greater than in the FRs for both clones (Table 2)
.
The VIPase activity of pooled IgG from C3H/gld mice was progressively inhibited by increasing concentrations of the serine protease inhibitor DFP (Fig. 4
A). Similarly, DFP inhibited the VIPase activity of IgG from a lupus patient (83±5% inhibition at 1 mM DFP; IgG code 523; data not shown). The biotinylated phosphonate diester Bt-Z, which is an affinity label for nucleophilic amino acids (20
, 24
, 25)
, formed irreversible adducts with C3H/gld IgG and lupus IgG, evident as 150 kDa bands on denaturing electrophoresis gels (Fig. 4B
). Reducing electrophoresis indicated that both subunits of C3H/gld IgG and lupus IgG were labeled with Bt-Z (heavy chains, 50 kDa; light chains, 25 kDa). Similarly, both Fv clones formed biotin-containing phosphorylated adducts that were stable to denaturing conditions. The formation of these adducts was inhibited by DFP. The adducts migrated with the expected mass of the 25 kDa monomer and an anomalous 50 kDa dimer (Fig. 4B
). Like the monomer adduct, the 50 kDa band was stainable with anti-c-myc antibody and it was reducible with ß-mercaptoethanol, indicating that it is an S-S bonded dimer (the Fv contains a 10 residue c-myc peptide tag). IgG and Fv samples heated at 80°C for 5 min, then allowed to bind Bt-Z, did not form adducts detectable by electrophoresis, indicating that covalent Bt-Z binding activity is dependent on the native structure of the proteins. These data are consistent with the presence of nucleophilic groups in the IgG and Fv preparations similar to those found in conventional serine proteases.
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Antibody selectivity
All [tyr10-125I]VIP cleavage reactions were conducted in the presence of excess albumin (15 µM), minimizing detection of nonspecific proteolytic activities. Inclusion of the alternate substrates eledoisin or bombesin (10 µM) in the reaction mixtures did not inhibit [tyr10-125I]VIP hydrolysis (<5% inhibition); whereas near-complete inhibition of [tyr10-125I]VIP cleavage was evident at an equivalent concentration of unlabeled VIP (91.9 ± 3.4% inhibition; data not shown; C3H/gld IgG 0.2 µM; cleavage of [tyr10-125I]VIP in the absence of alternate substrates, 48.0± 0.5% of available peptide). VIPase and Pro-Phe-Arg-MCA cleaving activities of IgG preparations from young and old C3H/gld mice were compared. Cleavage of the latter substrate at the amide bond linking Arg and the coumarin moiety serves as a measure of polyreactive proteolytic activity (12)
. IgG from aged C3H/gld mice showed elevated VIPase and reduced Pro-Phe-Arg-MCA cleaving activities compared with their young counterparts (Fig. 5
A, B). IgG from young mice displayed the opposite substrate preference, that is, greater Pro-Phe-Arg-MCA cleavage and lower [tyr10-125I]VIP cleavage. Previously we reported the thyroglobulin cleaving activity of the ATh and lupus IgG preparations described here (12)
. No correlation was evident between the levels of thyroglobulin and VIP cleavage by IgG preparations from 8 ATh and 10 lupus patients (r2=0.02;P=0.56, Fig. 5C
). Similarly, increased VIP cleavage by lupus IgG and ATh IgG preparations compared with healthy control IgG preparations was observed in the present study (Fig. 1)
, whereas decreased Pro-Phe-Arg-MCA cleavage in these disease groups has been reported previously (12)
. We concluded that the proteolytic activity of lupus and ATh IgG is selective for VIPase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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. Several previous studies have provided evidence linking proteolytic immunity and disease, including demonstration of alloantibody catalyzed factor VIII cleavage in hemophilia patients (15)
, thyroglobulin cleavage by spontaneously formed autoantibodies in lupus and ATh patients (12)
, and cleavage of certain model peptides in sera from rheumatoid arthritis patients (26)
.
The possibility of contamination was considered carefully. The presence of a generic protease contaminant in polyclonal IgG due to inefficient protein G affinity chromatography is inconsistent with the absence of VIPase activity in nonautoimmune IgG samples. Moreover, gel filtration studies of IgG from asthma patients purified as in the present study indicated VIPase activity to localize in a predictable manner in the 150 kDa intact IgG fraction, the 50 kDa Fab fragment prepared by papain digestion and the 25 kDa light chain fragment isolated by reduction and alkylation (22
, 27)
. Autoimmune sera may be hypothesized to contain anti-protease antibodies, which may lead to copurification of protease-containing immune complexes along with free IgG. However, this hypothesis is inconsistent with the following considerations. First, the VIPase activity was also observed in recombinant Fv clones from lupus patients subjected to selection by binding to VIP. Second, the VIPase activity of polyclonal IgG from serum was uncorrelated with the cleavage of other peptidic substrates (Pro-Phe-Arg-MCA and thyroglobulin), and additional peptides examined did not serve as alternate substrates for the VIPase activity. The selectivity for VIP is typical for antibody responses. Conventional proteases displaying selective VIP cleavage have not been described. Third, immunoadsorption studies failed to link the VIPase activity to contamination. Near-complete adsorption of the VIPase activity by immobilized anti-IgG was evident in concentrated as well as 100-fold diluted IgG solutions (1.22 µM and 12.2 nM IgG). This is inconsistent with the hypothetical antibody–contaminant complexes, because dilution of the IgG should induce dissociation of the complexes and reduced immunoadsorption of proteolytic activity; e.g., if the contaminant constitutes 2% of total protein and Kd for antibody-contaminant binding is 1 nM, diluting the IgG to 12.2. nM should result in adsorption of only 12% activity; computed from the equation Kd= [Ab] · [contaminant]/[Ab-contaminant complex].
The VIPase activity of IgG and Fv clones was inhibitable by the serine protease inhibitor DFP, which is consistent with previous mutagenesis studies indicating the presence of a serine protease-like catalytic triad in a model VIPase antibody (23)
. Furthermore, the VIPase IgG and Fv preparations displayed DFP-inhibitable covalent binding to a biotinylated phosphonate diester, an established probe for nucleophilic Ser residues of enzymes. Similar phosphonate esters have been used to select catalytic antibody fragments by phage display (20)
and map the location of the nucleophilic amino acid in an esterase antibody (28)
. As the VIPase Fv constructs are monoclonal, their ability to bind the phosphonate covalently suggests the serine protease-like mechanism of catalysis. In the case of the polyclonal VIPase samples, DFP inhibition of the proteolytic activity is the only direct indicator of mechanism. Covalent phosphonate binding by polyclonal IgG is consistent with but does not unambiguously establish a serine protease-like mechanism, as non-VIPase antibodies present in the polyclonal preparations may also express nucleophilic reactivities. Moreover, nucleophilic reactivity revealed from the covalent phosphonate binding studies is a necessary but not sufficient condition for catalysis. The presence of a nucleophile that is activated by interactions with neighboring residues (e.g., the hydrogen bonding network formed between the catalytic triad residues Ser-His-Asp in enzymes) is sufficient to explain covalent phosphonate binding, whereas a fully competent catalytic site must possess additional structural attributes allowing cleavage of the peptide bond, attack on the covalent reaction intermediate by a water molecule, and product release. Both subunits of polyclonal IgG (heavy and light chains) were labeled covalently by the phosphonate ester. The nucleophilic reactivity of antibody light chains has ample precedent (13
, 14
, 23
, 27)
and nucleophilic residues in heavy chains have also been identified (28
, 29)
.
The apparent Km values for polyclonal VIPase IgG preparations ranged from submicromolar to high micromolar values, indicating low to modest VIP binding strength compared with a high-affinity VIPase raised by experimental immunization (the latter displays nM Km; ref 23
; 1/Km equilibrium association constant). The Km values for all three lupus IgG samples studied (28–79 µM) suggests comparatively poor recognition of the ground state of VIP. Low-affinity antibodies are usually promiscuous in regard to recognition of other polypeptide antigens. As noted, this is not the case for the VIPase autoantibodies, including lupus IgG samples. The VIPase reaction was not impeded by polypeptides unrelated in sequence to VIP. Moreover, no correlation was evident between the VIPase activity and cleavage of a model peptide substrate or thyroglobulin. The specificity of VIP cleavage may reside therefore in chemical steps occurring after formation of the noncovalent antibody–antigen complex, i.e., nucleophilic recognition of the cleavage site in VIP within the context of noncovalent flanking residue recognition. Potential nucleophiles in Fv clones DM408 and DM506 have been identified by molecular modeling, as described previously (30)
. A Ser-His dyad is thought to be the minimal active site structure of serine transacylases (29)
. The VL domain of both clones contains a Ser residue in CDR2 (Kabat residues 50 and 52 in clone DM408 and DM506, respectively; Kabat numbering) that approaches a His residue in CDR1within hydrogen bonding distance (His34 and His31, respectively), which could allow acquisition of nucleophilic reactivity. The V regions of both clones express mutational patterns typical of antibodies subjected to B cell clonal selection, that is, replacement mutations that localize preferentially in the CDRs compared with the framework regions. Evidence that the serine protease activity is a heritable function encoded by antibody germline VL gene(s) has been reported elsewhere (31)
. A detailed discussion of the relationship between antibody chemical reactivity and clonal selection is beyond the scope of the present study, but the results suggest the existence of specific immunological triggers permitting recruitment and adaptive maturation of nucleophile-containing V genes encoding the VIPase activity.
The VIPase Fv clones were isolated from the lupus phage library by binding to immobilized VIP under conditions that minimize proteolysis. This explains the greater antigen binding strength of the Fv clones compared with polyclonal lupus IgG samples (100-fold and more, judged from 1/Km values). Presumably, higher Km VIPases are the majority species in polyclonal IgG, precluding identification of low Km autoantibodies in the catalysis assays. The Fv clones may not accurately model the bioactivity of the predominant VIPase species found in serum, i.e., the low-affinity catalysts, although they may still be useful as renewable VIPase sources for other types of structure–function studies. Another criticism of the Fv clones is that they might not contain natural VL/VH pairs (32)
. The use of large libraries offering a near-complete repertoire of combinatorial VL/VH diversity mitigates this objection somewhat. Four of 11 Fv clones selected by binding of the phages to immobilized VIP expressed VIPase activity. Catalysis by antibodies that bind the antigen strongly has been held to be a disfavored event, because stabilization of the antigen ground state might increase the activation barrier to reach the transition state (33)
. However, no anti-catalytic effects will occur if the stabilizing contacts in the ground state complex are preserved and additional stabilizing interactions develop with formation of the antigen–antibody transition state complex.
Identification of the peptide binds cleaved by the VIPases was not undertaken in the present study as extensive data are available concerning the scissile bond preferences of polyclonal and monoclonal VIPase antibodies (22
, 23)
. These antibodies were shown to cleave peptide bonds located between residues 7 and 22 of VIP. The autoantibodies are predicted to inactivate the peptide regardless of the cleavage site, as the entire sequence of VIP is required for binding to VIP receptors (34)
. A recent study describes a model VIPase light chain that cleaves VIP at Tyr20-Lys21 and Lys21-Lys22, inhibits VIP binding by high-affinity receptors in lung homogenates more potently than its catalytically deficient mutant, and blocks the smooth muscle relaxant effect of the peptide (2)
.
Precise details of how VIP regulates autoimmune responses are not understood, but it is clear that neuronally released VIP and VIP synthesized within T cells (4
, 35)
are important modulators of various immune responses. In addition to its anti-inflammatory effects mediated via receptors found on macrophages, mast cells, and other inflammatory cells (36)
, VIP regulates cytokine synthesis by CD4+ T cells (3
, 4)
and Fas ligand expression on T cells (5)
. In principle, all of the functions of extracellular VIP are susceptible to interference by the antibodies. As effective antagonists of VIP are not available, catalytic reagents such as the Fv clones reported here can be used to more precisely delineate the immunological roles of VIP. In view of ameliorative effects of exogenously administered VIP in an animal model of autoimmune disease (10)
, the endogenous peptide may mediate similarly favorable effects. The VIPases can be hypothesized therefore to induce amplified autoreactive immune responses. Relating VIPase molecular properties to clinical disease characteristics may yield clues about the functional role of VIPases in future studies. The number of IgG samples studied in the present report is too small to make firm conclusions about this point. For example, valuable guidance may be obtained from parameters such as the Km value. If turnover is assumed to be equivalent, the large Km values for VIPases from lupus IgG suggests that these can cleave the peptide appreciably only at high concentrations of the peptide, such as might accumulate close to the site of peptide release from neurons. In comparison, low Km VIPases may mediate important effects even at sites distant from neurons, where the VIP concentration is lower. Additional factors likely to govern the biological effects of VIPases include their ability to permeate organs in which VIP is a physiological mediator as well as their local synthesis by lymphocytes within the tissues.
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ACKNOWLEDGMENTS |
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Received for publication May 29, 2002. Accepted for publication November 27, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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) or a control noncatalytic IgG (
) (bottom). IgG, 0.2 µM. (tyr10-125I)VIP 0.4 nM, 20 h, 37°C. Elution with a gradient of acetonitrile in 0.1% TFA (20–40%, 5 min; 40–50%, 15 min; 0.5 mL/min).
