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Originally published In Press as doi:10.1074/jbc.M406719200 on July 21, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39611-39619, September 17, 2004
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Naturally Occurring Proteolytic Antibodies

SELECTIVE IMMUNOGLOBULIN M-CATALYZED HYDROLYSIS OF HIV gp120*

Sudhir Paul{ddagger}§, Sangeeta Karle{ddagger}, Stephanie Planque{ddagger}, Hiroaki Taguchi{ddagger}, Maria Salas¶, Yasuhiro Nishiyama{ddagger}, Beverly Handy||, Robert Hunter{ddagger}, Allen Edmundson¶, and Carl Hanson**

From the {ddagger}Chemical Immunology and Therapeutics Research Center, Department of Pathology and Laboratory Medicine, University of Texas–Houston Medical School, Houston, Texas 77030, the ||M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030, the Crystallography Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, and the **Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, California 94804

Received for publication, June 16, 2004 , and in revised form, July 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys432-Ala433 peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421–431 with a C-terminal amidino phosphonate diester mimetic of the Lys432-Ala433 bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
As the first class of Abs1 synthesized in the course of B cell development, IgM Abs often contain V domains that are close in sequence to germ line V genes. IgG Abs produced by differentiated B cells, in comparison, contain V domains that are more diversified by adaptive maturational processes occurring after exposure to the antigen. Recently, we reported that IgM µ subunits expressed as components of the B cell receptor account for the majority of the nucleophilic reactivity of the B cell surface, detected as the irreversible binding of haptenic covalently reactive antigen analogs (CRAs) containing an electrophilic phosphonate diester group (1). The phosphonate CRAs serve as probes for the activated nucleophiles found in non-Ab proteolytic enzymes (2) and proteolytic Abs (3). Secreted IgM Abs were reported to bind the CRAs irreversibly and catalyze the hydrolysis of model peptide substrates at rates exceeding those of IgG Abs (1). There was no expectation in these studies of selective catalytic cleavage of individual peptide antigens by the IgMs because no attempts were made to induce specific Ab responses by immunization procedures. Indeed, the proteolytic reaction was characterized by its promiscuity, in that tripeptide and tetrapeptide substrates with no apparent sequence similarity were cleaved, limited only by the requirement for a basic residue at the cleavage site (1). In contrast, in the case of specific IgG Abs directed to individual antigens, noncovalent Ab-antigen interactions guide the nucleophilic reaction and enable antigen-specific proteolysis, as judged from the selective covalent reaction of the Abs with polypeptide CRAs containing the phosphonate group within their antigenic epitopes (4).

Various innate and adaptive immune mechanisms have been documented to be important in defense against microbial infections. In the case of HIV-1, the coat protein gp120 is an important target of host immune responses. Oligomers of gp120 initiate viral infection by binding host cell CD4 receptors. In addition, monomer gp120 shed from viral and infected cell surfaces mediates certain toxic effects. Binding of gp120 to uninfected CD4+ T cells has been implicated in apoptotic depletion of these cells, a hallmark of AIDS (5), and the neurotoxic effect of free gp120 may contribute to dementia in HIV-infected subjects (6). gp120 is bound by certain Abs found in uninfected individuals (7), earning this protein the designation of a superantigen, i.e. an antigen recognized by Abs without the requirement for adaptive sequence diversification of the V domains. A weak correlation between IgG Abs that recognize gp120 as a superantigen and resistance to HIV infection has been noted (8). In infected subjects, gp120 induces the synthesis of Abs directed mainly to antigenic epitopes located in the mutable regions of gp120 (9). These Abs are generally ineffective against viral escape mutants appearing over the course of infection. Presently, no effective immunotherapeutic or vaccination strategies against HIV infection or soluble gp120 are available. Abs to the CD4 receptor binding site of gp120 developed by experimental immunization (10) and phage library protocols (11) have been proposed as immunotherapy candidates. Recently, immunization with the CRA derivative of gp120 was shown to induce the synthesis of specific IgG Abs that cleave gp120 (12). Proteolytic Abs offer the potential advantage of permanent inactivation of the target protein. Moreover, repeated reaction cycles mediated by a single catalyst molecule are predicted to inactivate multiple gp120 molecules. In comparison, conventional Abs bind gp120 stoichiometrically, and dissociation of the Ab-antigen complex releases the biologically active protein.

We report here the selective and efficient cleavage of the HIV coat protein gp120 by IgM Abs from uninfected humans and mice immunized with irrelevant antigens. Based on their reactivity with CRA probes, the IgMs appear to cleave gp120 via a nucleophilic mechanism. Selectivity for gp120 appears to be derived at least in part from recognition of a peptide determinant composed of gp120 residues 421–433, which is known to contribute contact sites for CD4 (13, 14). This determinant is also implicated in gp120 recognition as a superantigen by the conserved V domain regions of certain Abs (15, 16).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Polyclonal human sera were from subjects without evidence of infection or immunological disease (two females, three males; age 23–45 years). Monoclonal human IgM was from patients with Waldenström's macroglobulinemia diagnosed at the M. D. Anderson Cancer Center according accepted clinical criteria (bone marrow/lymph node biopsy, flow cytometry, serum immunoglobulin quantitation, and electrophoresis as appropriate). IgM concentrations in these samples as determined by rate nephelometry were 9.2–55.7 mg/ml. None of these patients had any known history of HIV infection. Murine serum Abs were from BALB/c mice (Harlan, Indianapolis, IN; pooled from 150 mice; age 8–12 weeks). Monoclonal murine IgMs were purchased as cell-free ascites fluid and are described by the supplier to be directed against certain major histocompatibility antigens (clones corresponding to catalog nos. 8702, 8704, 9008, 9010, and 9020; Cedarlane, Ontario, Canada). IgM in serum or ascites (1 ml) was purified by affinity chromatography on columns of immobilized Abs to human or murine IgM as in Ref 1, taking care that protein in the effluent had returned to undetectable levels prior to elution (A280 < 0.001) prior to elution with a pH 2.7 buffer. Further purification was on a Superose 6 FPLC gel filtration column (1) in two different solvents: 50 mM Tris-HCl, pH 7.7, 0.1 M glycine, 0.15 M NaCl, 0.025% Tween 20 (buffer B) or 6 M guanidine hydrochloride in buffer B adjusted to pH 6.5 with HCl (buffer C). Column calibration was with thyroglobulin (660 kDa), IgG (150 kDa), and albumin (67 kDa). The IgM eluted with an apparent molecular mass of 900 kDa close to the void volume of the column. IgM was renatured after buffer C chromatography by dialysis against buffer B (17). IgM Yvo, a monoclonal cryoglobulin, was purified by repetitive warming (37 °C) and cooling (4 °C; 3 cycles; Ref. 18) followed by affinity chromatography on the anti-human IgM column. IgM Yvo and the murine monoclonal IgM Abs contained {kappa} chains. IgG was purified on protein G-Sepharose columns (17) using as starting material the unbound fraction from the anti-IgM columns or cell-free ascites. Fab fragments were prepared by digesting 300 µl of IgM (1 mg/ml) with agarose-conjugated pepsin (0.6-ml gel, 30 min, 37 °C) in 100 mM sodium acetate, pH 4.5, 150 mM NaCl, 0.05% NaN3, 0.1 mM CHAPS) as recommended by the manufacturer (Pierce). The unbound fraction was dialyzed against buffer B, purified by FPLC gel filtration on a Superose 12 column and dialyzed against 50 mM Tris-HCl, pH 7.7, 0.1 M glycine, 0.1 mM CHAPS. Total protein was determined by the bicinchoninic acid method (Pierce). SDS-PAGE (4–20% gels) was conducted under reducing conditions (2-mercaptoethanol). Blots of the gels were stained with peroxidase-conjugated goat anti-human µ, {gamma}, {kappa}, and {lambda} Abs or goat anti-mouse µ, {gamma}, {kappa}, and {lambda} Abs followed by peroxidase-conjugated rabbit anti-goat IgG (Fc-specific) as in Ref. 17. Nominal molecular mass values were computed by comparison with standard proteins (14–94 kDa; Amersham Biosciences).

Proteolysis Assays and ELISA—Preparation of gp120, sEGFR, and BSA labeled with biotin at Lys residues (1–2 mol of biotin/mol of protein) is described in Refs. 4 and 12. Purified sCD4 (residues 1–183; NIH AIDS Reagent Program) and iodinated protein A (I-protein A) were biotinylated by similar methods (1.3 and 5.0 mol of biotin/mol of protein, respectively). I-protein A was obtained by treatment of protein A (Sigma) with iodine monochloride (Acros Organics, Inc.) to inactivate its Fc binding site (19). The gp120 (strain MN) is a recombinant protein expressed in the baculovirus system (Immunodiagnostics, Woburn, MA). Proteolysis assays were performed by incubating Bt-proteins with the Abs in 50 mM Tris-HCl, 100 mM glycine, pH 7.7, 1 mM CHAPS at 37 °C (12). The samples were boiled in buffer containing SDS and 2-mercaptoethanol and electrophoresed on SDS-gels. Cleavage was determined by densitometry of electroblots stained with streptavidin-peroxidase. Kinetic parameters were determined by fitting rate data at varying Bt-gp120 concentrations to the quadratic equation 1 (20)

(Eq. 1)
where [Ct] and [St] are the total concentrations of catalyst and substrate, and [CS] is the concentration of the catalyst-substrate complex. The gp120pep-CRA (Bt-KQIINMWQEVGN with the amidino phosphonate diester group at the C terminus) was analyzed as an inhibitor of proteolysis. Stock gp120pep-CRA solutions were in ethanol (final ethanol concentration in proteolysis assay, 6%). Purified porcine pepsin used as control in Fab cleavage studies was from Sigma. N-terminal sequencing of gp120 fragments electroblotted from electrophoresis gels was performed as in Ref. 20 (Applied Biosystems model 492 Procise cLC sequencer). Cleavage of the amide bond linking aminomethylcoumarin to the C-terminal amino acid in Boc-Glu-Ala-Arg-AMC (Peptide International, Louisville, KY) was measured as in Ref. 1 by fluorometry ({lambda}ex 360 nm, {lambda}em 470 nm) with authentic AMC as the standard curve. Kinetic parameters were obtained by fitting rate data at increasing peptide-AMC concentrations to the Michaelis-Menten-Henri equation.

(Eq. 2)

ELISA for VH3+ IgM Abs was done using I-protein A-immobilized 96-well plates (500 µg/well) as in Ref. 21. The wells were treated with 5 µg/ml IgM samples in 10 mM sodium phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 containing 0.1% skim milk. Detection was with peroxidase-conjugated goat anti-human IgM (1:1,000; Sigma) (8).

Irreversible CRA Binding—Synthesis of the biotin-containing hapten phosphonate CRA and the gp120pep-CRA and their irreversible reaction with proteases and Abs has been described previously (4, 22, 23). Formation of CRA-IgM adducts was measured by reducing SDS-electrophoresis, electroblotting, and densitometry using a streptavidin-peroxidase conjugate (4). Band intensities are expressed in arbitrary area units. Initial velocities were computed as slopes of progress curves (incubation for 20, 40, 60, 120 and 220 min; r2 > 0.9 for all data reported here).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Catalytic Activity of Polyclonal IgM Abs—Each IgM preparation purified from five human sera expressed readily detectable ability to cleave biotinylated gp120 (Bt-gp120) (Fig. 1A), assessed by depletion of the parent gp120 band and appearance of fragments with lower mass in electrophoresis gels (Fig. 1B; the recombinant protein migrates with nominal molecular mass of ~95 kDa, presumably because of incomplete glycosylation in the baculovirus expression system). With increasing length of incubation, accumulation of the 50-kDa biotin-containing fragment and depletion of the 80-kDa fragment were evident, suggesting that the latter product is susceptible to further hydrolysis. IgG samples from these sera did not cleave gp120 detectably. The data in Fig. 1A are expressed per equivalent combining sites of IgM and IgG (10 and 2, respectively; note, however, that all 10 IgM valences are usually not filled; Ref. 24). Superior IgM catalysis, therefore, cannot be ascribed to the greater number of IgM combining sites. Essentially identical results were obtained using IgM and IgG Abs prepared from the pooled sera of immunologically unstimulated BALB/c mice (87.9% cleavage/20 h/150 nM IgM combining sites; undetectable gp120 cleavage at equivalent IgG combining site concentration; reaction conditions as in Fig. 1A).



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  		FIG. 1.
Cleavage of Bt-gp120 by polyclonal human IgM and IgG preparations. A, scatter plot of gp120 cleaving activity of IgM and IgG Ab fractions from five healthy humans. The Ab combining site concentration was 150 nM (decavalent IgM, 15 nM; bivalent IgG, 75 nM). Reaction conditions: 20 h, 37 °C, 100 nM Bt-gp120. Solid lines are the means (IgM, 53.3 ± 25.4%; cleavage by IgG is below detection limit (<5%)). Inset, typical reducing SDS-PAGE (4–20% gels) results showing human serum IgM purified by affinity chromatography on immobilized anti-IgM Ab and stained with Coomassie Blue (lane 1) and peroxidase-conjugated Abs to human µ chains (lane 2), {kappa} chains (lane 3), and {lambda} chains (lane 4). B, streptavidin-peroxidase-stained reducing SDS-gel lanes showing time-dependent cleavage of Bt-gp120 by pooled polyclonal human IgM. BC, Bt-gp120 incubated for 23 h in the absence of Abs. IgM, 50 nM; Bt-gp120, 100 nM. C, pooled human serum IgM purified by affinity chromatography on immobilized anti-µ Abs was subjected to two sequential cycles of denaturing gel filtration (broken line, cycle 1; solid line, cycle 2; Superose 12 column) in 6 M guanidine hydrochloride. Inset, streptavidin-peroxidase-stained SDS-gel lanes with evident cleavage of 100 nM Bt-gp120 by 50 nM IgM obtained from the second cycle of denaturing gel filtration (lane 2) and an equivalent concentration of the control IgM analyzed without denaturation (lane 3). Reaction time, 16 h. Lane 1, Bt-gp120 incubated with diluent instead of IgM.

 
Next, we examined the possibility of non-IgM proteases as the explanation for the observed catalytic activity. In particular, there is the risk of copurification of contaminants bound noncovalently to the IgM. A denaturing solvent (6 M guanidine hydrochloride) was employed to ensure dissociation of potential noncovalent complexes in an IgM preparation purified previously by affinity chromatography using immobilized anti-IgM Abs. The IgM was then subjected to two sequential cycles of FPLC gel filtration in the denaturing solvent (Fig. 1C). The 900-kDa IgM fraction recovered by this procedure was renatured by dialysis, and the catalytic activity was measured. The renatured IgM displayed Bt-gp120 cleaving activity in the same range as the starting IgM fraction (inset, Fig. 1C; 82.2 ± 9.2 and 78.8 ± 1.0% cleavage, respectively). To our knowledge, no serum proteases with mass corresponding to the 900-kDa IgM fraction are described in the literature. Because low molecular mass proteins are well separated from the 900-kDa IgM fraction by the denaturing gel filtration procedure, we are unable to explain the observed gp120 cleavage as being the result of trace contaminants.

Catalytic Activity of Monoclonal IgM Abs—Next, we studied the gp120ase activity of monoclonal human and murine IgM Abs. All but one of the IgMs (IgM Yvo) were prepared by an identical affinity chromatography procedure using immobilized anti-IgM Abs. IgM Yvo, a cryoglobulin, was purified by three cycles of precipitation prior to the affinity chromatography step. 10 of 12 monoclonal IgMs from patients with Waldenström's macroglobulinemia (including IgM Yvo) and four of five murine monoclonal IgMs displayed the gp120 cleaving activity (>10% consumption of available gp120; Fig. 2A). The monoclonal IgMs from five patients with Waldenström's macroglobulinemia were purified in two independent experiments, and their gp120 cleaving activities were measured. The interexperiment variations of the activity were within the range of experimental error of the cleavage assay (6.8 ± 4.2%; n = 5). This suggested that the differing activities of the individual monoclonal IgMs in Fig. 2A are not caused by variables associated with the purification method.



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  		FIG. 2.
gp120 cleavage by monoclonal human and murine IgM antibodies. A, VH3+ ({circ}) and non-VH3 () human monoclonal IgMs from patients with Waldenström's macroglobulinemia; {square}, murine IgMs. Bt-gp120, 100 nM; IgM, 50 nM, 17h, Data were obtained by densitometry of streptavidin-peroxidase-stained reducing SDS-gels. Inset, SDS-gel showing IgM Yvo stained with Coomassie Blue (lane 1), anti-human µ chain Ab (lane 2), and anti-{kappa} chain Ab (lane 3). B, streptavidin-stained blots showing Bt-gp120 or Bt-I-protein A (100 nM) incubated in the absence and presence of human monoclonal IgM (code 1812; 50 nM, 17 h).

 
A subset of IgMs containing VH3 family VH domain from uninfected humans has been reported previously to bind gp120 (7, 15, 16). VH3+ IgMs can be identified by screening for binding of iodinated protein A because the iodination reaction inactivates the Fc binding site of protein A while leaving intact the recognition of this protein as a superantigen by the V domains (19). In the present study, 5 VH3+ monoclonal IgMs from patients with Waldenström's macroglobulinemia were identified by measuring the binding of I-protein A by ELISA (n = 12 IgMs; A490 range 1.52–2.81, S.D. values < 12% of the mean in each case; the remaining 7 IgMs studied did not bind I-protein A detectably, A490 < 0.11). All but one of the IgMs with I-protein A binding activity and all but one of the IgMs devoid of this activity were observed to cleave gp120 (Fig. 2A). To the extent that the absence of detectable I-protein A binding activity reflects the usage of non-VH3 family domains, these results suggest that the proteolytic activity is not restricted to VH3+ IgMs. This is supported by the observed gp120 cleavage by IgM Yvo, which contains a VH2 family VH domain (18; as expected, IgM YVO was devoid of I-protein A binding activity determined by ELISA). There was no evident association between the extent of gp120 cleavage and I-protein A binding activity within the group of VH3+ IgMs (n = 5, r = 0.05, p = 0.72). Importantly, none of the five VH3+ IgMs cleaved I-protein A detectably (an example is shown in Fig. 2B). Taken together, these results suggest that the ability to cleave gp120 is a distinctive property of individual IgM Abs, as opposed to a generalized consequence of superantigen recognition.

IgM Yvo, which displayed the greatest gp120 hydrolyzing activity among the 12 Waldenström's IgMs studied, was assayed at two stages in its purification process, i.e. after three cycles of precipitation induced by warming and cooling, and after the additional affinity chromatography step using an anti-IgM column. The levels of gp120 cleavage before and after the affinity chromatography step were essentially identical (46.4 ± 0.6% (S.D.) and 44.3 ± 4.9% Bt-gp120 cleaved/50 nM IgM/6 h, respectively; reactions as in Fig. 2A). Purification of the IgM preparation to constant specific activity supports assignment of the Bt-gp120 cleaving activity to the Abs. The possibility of contamination was studied further using the Fab fragment prepared by gel filtration after digestion of IgM Yvo with immobilized pepsin. Concentration-dependent Bt-gp120 cleavage by the 50–55-kDa Fab fraction was evident (Fig. 3B). Undigested IgM subjected to gel filtration under identical conditions eluted as the 900 kDa peak, with no gp120 cleaving activity detected in fractions corresponding to the 50–55-kDa mass range. Next, we considered the possibility that pepsin released from the immobilized pepsin column may be responsible for the observed activity. The pH optimum of pepsin is 1.5–2.7 depending on the substrate, and the enzyme is inactivated irreversibly at the neutral pH employed for Bt-gp120 hydrolysis assays (25). Consistent with the reported properties of pepsin, purified pepsin at concentrations as large as 1.2 µM did not cleave Bt-gp120 detectably (not shown; reaction conditions as in Fig. 3B), suggesting that the proteolytic activity of the Fab cannot be explained by pepsin contamination.



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  		FIG. 3.
Bt-gp120 cleavage by IgM Fab fragment. A, gel filtration profile (Superose 12) of IgM Yvo without (broken line) and with (solid line) digestion with immobilized pepsin. Inset, silver-stained nonreducing (lane 1) and reducing (lane 2) SDS-gels of the 55-kDa Fab fragments. The higher and lower molecular mass fragments in the reducing gel correspond to the Fab heavy chain fragment and light chain component. B, streptavidin-peroxidase-stained SDS-gels showing cleavage of 0.1 µM Bt-gp120 incubated with increasing concentrations of Fab Yvo for 48 h.

 
To identify peptide bonds in gp120 susceptible to cleavage by IgM Yvo, nonbiotinylated gp120 at a concentration in excess of the IgM was employed as substrate (Fig. 4). Several Coomassie Blue-stained bands attributable to cleavage by the IgM were evident (smeared products at nominal mass range 80 kDa, 50 kDa, and more well defined bands at 15.4–18.8 kDa; the bands at 25 and 70 kDa correspond to IgM Yvo light and heavy chains, respectively; Fig. 4). Possible reasons for the difference in Bt-gp120 (Figs. 1 and 3) and nonbiotinylated gp120 cleavage patterns include (a) the biotinylation reaction at Lys residues and associated experimental manipulations could alter the substrate properties of the protein; (b) the biotin content of the Bt-gp120 was small (1.4 mol/mol), with the result that certain Coomassie Blue-stainable fragments may not contain biotin, and the profile of biotinylated products may not accurately predict their relative abundance; and (c) under the conditions of near complete gp120 cleavage in Fig. 4, the products may be digested further by the catalyst, allowing increased accumulation of the lower mass bands and decreased accumulation of the intermediate sized products. The 50-kDa fragment in Fig. 4 was not present in sufficient amounts for further analysis. The remaining fragments bands were subjected to N-terminal amino acid sequencing (Table I). The 80-kDa gel region yielded a sequence corresponding to the N terminus of gp120. Because no internal gp120 peptide sequence was detected, the cleavage site could not be identified. However, from the mass of this N-terminal fragment (80 kDa), it may be concluded that the cleavage site is located in the C-terminal half of gp120. gp120 fragments with N-terminal sequences corresponding to gp120 residues 433–442 were identified in the gel regions corresponding to 15.4–16.4-kDa and 17.6-kDa gel regions, permitting unambiguous identification of the 432–433 peptide bond as one of the cleavage sites. In addition, two fragments with N-terminal sequences corresponding to gp120 residues 1–10 were present in the 15.4–16.4-kDa and 18.8-kDa gel regions. These fragments suggest the existence of two additional cleavage sites located in the N-terminal half of the protein.



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  		FIG. 4.
Coomassie Blue-stained blots of SDS-gels showing gp120 cleavage by IgM Yvo. Shown are 8.5 µM gp120 with IgM Yvo (50 nM) (lane 1), IgM Yvo alone (lane 2), and gp120 alone (lane 3) incubated for 46 h. The 70 kDa band (visible at the bottom of the smear labeled 80 kDa) and the 25 kDa band in lane 1 correspond to IgM heavy and light chains, respectively. Blot regions corresponding to 15.4–16.4, 17.6, 18, and 80 kDa were subjected to N-terminal sequencing in Table I.

 


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  		TABLE I
N terminal sequences (10 residues) of gp120 fragments generated by IgM Yvo

Reaction conditions: 8.5 µM gp120, 50 nM IgM, 46 h. N.I., not identified; however, identification of the 15.4–16.4- and 18.8-kDa fragments with N termini corresponding to gp120 residues 1–10 indicates two cleavage sites located in the N-terminal half of the protein. X, unidentified amino acid.

 
Kinetics and Substrate Selectivity—Treatment of Bt-BSA, Bt-sCD4, or Bt-sEGFR with polyclonal human IgM did not result in noticeable depletion of electrophoresis bands corresponding to the full-length form of these proteins (Fig. 5). Under these assay conditions, readily detectable Bt-gp120 cleavage was observed. Similarly, there was no detectable depletion of full-length Bt-sCD4, Bt-sEGFR, and Bt-BSA bands treated with IgM Yvo and polyclonal murine IgM (reaction conditions as in Fig. 5; cleavage of Bt-gp120 by these Ab preparations, 69.1 and 97.4%, respectively).



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  		FIG. 5.
Selectivity of gp120 cleavage. Streptavidin-peroxidasestained reducing SDS-polyacrylamide gels showing Bt-gp120, Bt-sEGFR, Bt-BSA, and Bt-sCD4 incubated for 22 h in diluent or polyclonal human IgM (50 nM). Bt-protein, 0.1 µM.

 
The kinetic parameters for polyclonal human IgM, monoclonal human IgM Yvo, and monoclonal murine IgM 8704 were determined by fitting the observed initial rates at increasing concentrations of gp120 to the general quadratic equation predicting the concentration of the catalyst-substrate complex (Table II). The method is applicable to the study of the reaction kinetics when the gp120 concentrations are lower than the Km. The apparent Km is the value yielding the best fit observed velocity plotted versus a series of assumed [IgM-gp120] concentrations, and apparent kcat is the slope of the velocity versus [IgM-gp120] plot. Correlation coefficients for the plots corresponding to the data in Table II were >0.9. Observed kcat values were as great as 2.1/min and varied over a 52.5-fold range, and observed Km values were as low as 1.3 µM and varied over a 23.8-fold range. These data support our conclusion that different IgMs recognize gp120 with distinct properties.


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  		TABLE II
Apparent kinetic parameters for IgM catalyzed biotinylated gp120 cleavage

Substrates are indicated in parentheses. Increasing gp120 concentrations (2, 1, 0.5, 0.25, and 0.125 µM) were treated in duplicate with 50 nM IgM for a sufficient length of time to yield gp120 cleavage levels in the measurable, linear phase of the reaction. For method of kcat and Km computation, see "Results." Kinetics of Glu-Ala-Arg-AMC was determined as in Ref 1.

 
Some of the IgM preparations examined in the present study were recently described to hydrolyze small peptidyl methylcoumarinamide substrates (1). In the present study, we compared the kinetics of IgM Yvo-catalyzed cleavage of Bt-gp120 and Glu-Ala-Arg-AMC. The sequence of gp120 examined in the present study (MN strain) does not contain the tripeptide determinant Glu-Ala-Arg. The Km for gp120 cleavage was 300-fold smaller than for Glu-Ala-Arg-AMC (Table II). Because the Km is an approximate measure of initial substrate recognition, the results suggest that the IgM binds gp120 with superior affinity compared with the irrelevant peptide substrate.

Selective Nucleophilic Reactivity—The synthetic peptide analog of gp120 residues 421–432 containing phosphonate diester and biotin groups (gp120pep-CRA; top structure, Fig. 6A) was developed previously as a covalent probe for nucleophilic Abs that recognize this region of gp120 (4, 22). The control hapten CRA contains an identical phosphonate diester group but is devoid of the peptide determinant. The phosphonate diester group is known to inhibit enzymes that utilize the serine protease mechanism for peptide bond cleavage (26, 27). Synthetic peptides spanning gp120 residues 421–432 are shown to be components of the gp120 superantigenic site recognized noncovalently by IgMs (15, 16). The gp120pep-CRA was reported to react irreversibly with Abs raised by immunization with a synthetic gp120 peptide corresponding to residues 421–436 (4), suggesting the validity of this compound as a probe for specific Abs to gp120. In the present study, progressive inhibition of the cleavage of Bt-gp120 by IgM Yvo was observed at increasing gp120pep-CRA concentrations (Fig. 6B). Covalent gp120pep-CRA binding by the IgMs was measured by estimating the biotin content in protein adduct bands on denaturing electophoresis gels. All six IgM Ab preparations studied (four human monoclonal IgMs, one murine monoclonal IgM, and one human polyclonal IgM preparation) formed covalent adducts with the gp120pep-CRA. For every IgM preparation, the rates of adduct formation with the gp120pep-CRA were superior to the adducts formed by hapten CRA I, which is devoid of the gp120 peptide sequence (Fig. 7A; p = 0.009, Student's t test, two-tailed). The µ chain subunit accounted for the majority of covalent gp120pep-CRA binding except in the case of monoclonal IgM Yvo, in which the light chain subunit was the predominant reaction site (Fig. 7B). The CRA adducts accumulated linearly as a function of time (e.g. IgM Yvo light chain adducts shown in Fig. 7C). Formation of the gp120pep-CRA adducts by the IgMs was inhibited in the presence of synthetic gp120(421–436) devoid of the phosphonate group (Fig. 7D). These observations suggest a nucleophilic mechanism of catalysis in which noncovalent recognition of gp120 residues 421–432 may contribute to the observed selectivity for gp120.



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  		FIG. 6.
gp120(421–431)-CRA inhibition of Yvo IgM gp120ase activity. A, phosphonate diester analog of gp120 residues 421–433 (gp120pep-CRA) and the haptenic phosphonate diester devoid of the gp120 peptide sequence (hapten CRA). B, inhibition of IgM Yvo (50 nM) catalyzed Bt-gp120 (0.1 µM) by gp120pep-CRA. Incubation was for 15 h. Inset, streptavidin-peroxidase-stained SDS-gels showing Bt-gp120 incubated with IgM Yvo in the absence (lane 2) and presence of 10 µM gp20pep-CRA lane 3). Lane 1, control Bt-gp120 incubated in diluent.

 



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  		FIG. 7.
Irreversible gp120pep-CRA binding by IgM. A, initial velocities for formation of hapten CRA adducts and gp120pep-CRA adducts by human monoclonal IgMs ({circ}, VH3+ IgM, codes 1801, 1816, 1818; , VH2+ Yvo IgM), monoclonal murine IgM ({square}; clone 8704), and polyclonal human IgM ({blacksquare}). Initial velocities were computed as the intensities of the CRA-containing bands measured in duplicate and represent the sums of intensities of the H chain-CRA and L chain-CRA bands. AAU, arbitrary area units. Reaction conditions: IgM, 150 nM; hapten CRA or gp120pep-CRA, 10 µM. Inset, streptavidin-peroxidase-stained reducing SDS-gels showing adducts of gp120pep-CRA (lane 1) and hapten CRA (lane 2) formed by IgM code 1801. B, initial velocities for formation gp120pep-CRA adducts by µ chains and {kappa}/{lambda} chains of human monoclonal IgMs. Symbols and reaction conditions are as in A. C, example of progress curves used for computing initial velocities. Shown are accumulation of gp120pep-CRA adducts and hapten CRA adducts with the L chain of IgM Yvo. Reactions are as in A. Inset, cut-outs of the L chain adduct bands from streptavidin-peroxidase-stained SDS-gels corresponding from left to right to progressively increasing times of incubation shown in the curves. D, streptavidin-peroxidase-stained reducing SDS-gels showing adducts of gp120pep-CRA formed by polyclonal human IgM in the absence (lane 1) and presence of synthetic gp20(421–436) (500 µM, lane 2) and by IgM Yvo in the absence (lane 3) and presence of synthetic gp20(421–436) (500 µM, lane 4). Reaction conditions are as in A. Incubation was for 4.5 h. In addition to the 70-kDa µ chain band and the 25-kDa {kappa}/{lambda} band, a 50 kDa band is evident in lane 1, identified previously as a µ chain fragment based on staining with anti-µ antibody (1).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These studies indicate the ability of IgM Abs to catalyze the cleavage of the HIV coat protein gp120 selectively. The Abs were from uninfected humans and immunologically naïve mice. IgG Abs, products of B cells at more advanced stages of differentiation, did not cleave gp120 appreciably. The observed selectivity of IgM-catalyzed gp120 hydrolysis, therefore, appears to be an intrinsic property of the preimmune Ab repertoire, as opposed to the Ab response mounted by adaptively matured B cells. Reactions such as esterolysis and hydrogen peroxide synthesis are also catalyzed by certain Abs regardless of the adaptive maturational status of the V domains (28, 29). Evidence for superior catalysis by IgMs compared with IgGs has been reported previously using small model peptide substrates (1), but the anti-viral catalytic selectivity of IgMs from uninfected donors observed in the present study is a novel finding to our knowledge.

Identically purified monoclonal IgMs expressed differing levels of gp120 cleaving activity as shown in Fig. 2A. Similarly, formation of covalent CRA adducts with different IgMs proceeded at differing rates, and the preferred subunit at which the reaction occurred was not identical for different IgMs. As the constant domain architecture of the IgMs is conserved, the results suggest that the catalytic activity is a V domain property. The phosphonate diester group of the CRA probe inhibits serine proteases irreversibly by covalent binding at activated Ser nucleophile (26, 27). The reaction with the CRAs was detected for every IgM preparation studied, suggesting the nucleophilic mechanism as a shared property of the catalysts. Previously, the presence of a serine protease-like catalytic triad has been suggested from site-directed mutagenesis studies of a germ line configuration light chain (30). IgG Abs in the preimmune murine and human repertoires are also reported to express promiscuous proteolytic activities, albeit at levels lower than observed using the IgMs (1, 17, 31). The broadly distributed gp120 cleaving activity of IgMs described here is consistent with the germ line origin of the catalytic activity.

Even if the intrinsic affinity of the individual combining sites is small, avidity effects caused by the decavalent character of IgM could strengthen the binding to antigens expressing repeat epitopes. However, the possibility of multivalent IgM binding to the same gp120 molecule is not favored at gp120 concentrations in excess of IgM in the solution phase reactions employed in the present study. Moreover, gp120 does not contain known repeat epitopes. These arguments suggest that the superior catalytic activity of IgMs compared with IgGs does not derive from avidity effects. Positive cooperativity effects such as those described for the independent binding of different antigen molecules by bivalent IgGs (32) could theoretically furnish favorable contributions in catalysis. The sequence of events as individual IgM combining sites bind antigen has not been elucidated, but previous studies have suggested that only 5 of the 10 IgM combining sites are occupied at excess antigen concentration (24), arguing against the possibility of positive cooperativity.

Catalysis by adaptively diversified IgG Abs may be disfavored because of the opposing requirements for efficient catalysis and B cell differentiation processes. Occupancy of the B cell receptor complex (membrane-bound Ig subunits together with noncovalently associated signal transducing proteins) by the antigen drives B cell division. Efficient catalysis, on the other hand, requires release of the product antigen fragments, which is predicted to result in reduced B cell receptor occupancy and cessation of antigen-driven B cell clonal selection. A plausible explanation for the results reported here, therefore, is that the proteolytic activity is lost attendant to V domain somatic diversification after the B cells switch from production of the IgM class to IgG class Abs. Alternatively, the unique constant domain architecture of IgM may contribute to maintaining the integrity of the catalytic site, in which case isotype switching itself is predicted to result in reduced catalytic activity. These explanations are not mutually exclusive. Both explanations are consistent with the hypothesis of disfavored proteolysis in the later stages of B cell differentiation. The latter explanation is supported by the results of monovalent Fab studies. The Fab fragment of a monoclonal IgM expressed the gp120 cleaving activity, consistent with assignment of the activity to the V domains, but the Fab preparations reproducibly displayed ~30-fold lower activity than computed for the individual combining sites of pentameric IgM. Disruption of the constant domain architecture of IgM, therefore, may be deleterious for catalysis. Pepsin employed to prepare the Fab cleaves µ chains on the C-terminal side of the CH2 domain (33), which is noted for its conformational flexibility (34). Altered levels of antigen binding by the same V domains expressed as full-length IgG Abs containing constant domains belonging to different isotypes are reported (e.g. 35), but the possibility of allosteric constant domain regulation of antigen recognition in IgMs has not been studied to our knowledge.

An intriguing aspect of the present study is the evident selectivity of the catalytic IgMs for gp120. The selectivity cannot arise from the local chemical interactions at dipeptide units because the same dipeptide units are present in other poorly cleaved proteins. Adaptively matured Abs obtained by experimental immunization are shown to express antigen-selective proteolytic activity attributable to noncovalent recognition of individual epitopes (12, 20). A role for noncovalent gp120 recognition is supported by the comparatively small Km for IgM-catalyzed hydrolysis of this protein. These values are 1–2 orders of magnitude lower than the apparent Km for promiscuous proteolytic reactions catalyzed by the IgMs (Table II; also see Ref. 1). Moreover the peptidyl CRA analog of gp120 (residues 421–431 with an amidino phosphonate mimetic of residues 432 and 433 at the C terminus) reacted with the IgMs more rapidly than the hapten CRA devoid of the gp120 peptide, and irreversible complexation of the peptide-CRA was inhibited by the synthetic gp120 peptide devoid of the phosphonate diester group. This reactivity pattern suggests that noncovalent recognition of the peptide component facilitates the nucleophilic attack on susceptible electrophilic groups by the Abs.

Superantigens bind Abs found in the preimmune repertoire by contacts at conserved regions encoded by germ line V genes. The superantigenic character of gp120 derives from the recognition of discontinuous gp120 peptide segments, including the segment composed of residues 421–433 (15, 16). As noted above, the importance of this peptide segment in IgM-catalyzed gp120 hydrolysis is suggested by the selective covalent binding of the peptide CRA. IgM Yvo cleaved the peptide bond linking gp120 residues 432 and 433, which is also located within this peptide region. Our model of IgM-catalyzed gp120 hydrolysis entailing noncovalent recognition of gp120 as a superantigen is analogous to the mechanism utilized by specific catalytic IgG Abs (20), except that the noncovalent interactions are dominated by contacts at conserved V domain regions instead of the adaptively mutated regions. Certain aspects of the model remain to be verified. For instance, the identity of the nucleophiles and the relative contributions of the light and heavy chains in the catalytic reaction remain to be determined. Adducts of gp120pep-CRA were formed preferentially at the heavy chain of five IgM preparations and at the light chain of one IgM preparation. Previous reports indicate that the light and heavy chains can each express catalytic nucleophiles (36–38). Another interesting property of the monoclonal IgMs is their ability to cleave multiple peptide bonds in gp120, analogous to the reaction profiles of monoclonal Ab light chain catalyzed hydrolysis of gp41 (38) and vasoactive intestinal peptide (20). As suggested previously (12), such fragmentation profiles may be explained by the formation of alternate Ab-gp120 ground state complexes with different peptide bonds positioned in register with the nucleophilic residue. When the Ab recognizes a conformational epitope, the alternate cleavage sites must be spatially adjacent, but they can be distant in the linear sequence, producing a complex fragmentation pattern.

Noncovalent binding of gp120 as a superantigen has been reported as a property of VH3+ Abs (21, 39, 40). VH3+ IgMs can conveniently be identified by binding to iodinated protein A, the archetypical B cell superantigen (19). In the present study, the IgMs did not cleave I-protein A detectably. No relationship between the cleavage of gp120 and binding of I-protein A by the IgMs was evident. Moreover, IgM Yvo, which contains a VH2 family VH domain (determined from the amino acid sequence reported in Ref. 18), displayed the gp120 cleaving activity. It may be concluded that the catalytic activity is not restricted to VH3+ IgMs. The cleavage of gp120, therefore, does not appear to be the generalized consequence of superantigen recognition by VH3+ IgMs. Additional relevant considerations are: (a) because of rapid product release, catalytic IgMs will not form stable complexes with gp120 and will be difficult to measure by assays relying on detection of noncovalent gp120-IgM complexes; and (b) if the appropriate assays are employed, the turnover capability allows more sensitive detection of the catalysts compared with noncatalytic IgMs. At the IgM (15 nM) and gp120 concentrations (100 nM) in Fig. 1A, a noncatalytic Ab with Kd 31 µM will bind only 0.5 nM gp120 at equilibrium (computed from Equation 3,

(Eq. 3)
where [Ab0]and [Ag0] are Ab and antigen concentrations at time 0). In comparison, 70 nM gp120 will be cleaved over 20 h under similar conditions by a catalytic IgM preparation with kcat 2.1/min and Kd equivalent to the noncatalytic Ab cited above (computed as,

(Eq. 4)
where Pt is product concentration at time t and k is kcat/Km; Ref. 41).

Catalytic Abs produced spontaneously by the immune system have been viewed until now as pathogenic effector molecules, e.g. catalytic autoantibodies directed to vasoactive intestinal peptide (42) and nucleic acids (43) and the catalytic antibodies to Factor VIII in hemophilia patients undergoing Factor VIII replacement therapy (44). The present study suggests that catalytic immune responses could also fulfill a protective role in certain circumstances. Apoptosis of neurons and T lymphocytes induced by monomeric gp120 has been implicated, respectively, in the dementia (6) and decline of CD4+ T cells occurring in AIDS patients (5). The IgM-catalyzed cleavage of monomeric gp120 shed from HIV, therefore, could potentially exert beneficial effects. A caveat is the possibility of inhibition by naturally occurring serine protease inhibitors. In the absence of inhibitors, blood-borne IgM found in humans at 2 mg/ml in blood may be computed to hydrolyze 50 and 90% of gp120 present at concentrations «Kd in 4.6 min and 15.5 min, respectively (assuming Kd 31 µM, kcat 2.1/min, Table II). Similarly, if cleavage of trimeric gp120 found as a complex with gp41 on the HIV surface proceeds at the rate observed for the monomeric protein, only short time periods are needed to hydrolyze the majority of viral gp120 (gp120 concentrations in infection remain « observed K; e.g. 106d HIV copies/ml with 100 gp120 molecules/virion correspond to ~2 x 10–13 M gp120; Ref. 45). The recognition of gp120 residues 421–433 supports a protective role for IgM Abs because this region of gp120 also contributes certain amino acids important in host cell CD4 binding (13, 14). Fragments generated by cleavage at the IgM-sensitive Lys432-Ala433 bond are reportedly devoid of CD4 binding activity (46). IgG Abs that bind the gp120 superantigenic site noncovalently are described as resistance factors to HIV infection (8). Initial studies conducted as in Ref. 47 suggest that polyclonal human IgM can neutralize the infection of peripheral blood mononuclear cells by primary HIV-1 isolates under low serum conditions.2 Previously, Berberian et al. have discussed their unpublished studies suggesting HIV neutralization by Abs from uninfected subjects in the absence of serum (Note 11 in Ref. 7).

Specific targeting of viral proteins has been a long-standing goal in catalytic Ab research. The results of the present study may be relevant to HIV vaccine design. Synthetic peptides containing gp120 residues 421–433 have been advanced as vaccine candidates (48, 49) in part because these residues are comparatively conserved in diverse HIV strains. The gp120 peptidyl CRA described here is a potential immunogen for induction of proteolytic Abs with strengthened recognition of the gp120 superantigenic site. A CRA derivative of full-length gp120 induces the synthesis of catalytic Abs (12), but Abs to irrelevant epitopes probably dominate the response to this immunogen.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI31268 and AI46029. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Chemical Immunology and Therapeutics Research Center, University of Texas Health Science Center, 6431 Fannin, Medical Science Bld., Rm. 2.250, Houston, TX 77030. Fax: 713-500-0574; E-mail: Sudhir.Paul{at}uth.tmc.edu.

1 The abbreviations used are: Ab, antibody; AMC, 7-amino-4-methylcoumarin; BSA, bovine serum albumin; Bt-, biotinylated; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CRA, covalently reactive analog; ELISA, enzyme-linked immunosorbent assay; Fab, fragment antigen binding; FPLC, fast protein liquid chromatography; HIV-1, human immunodeficiency virus type 1; I-protein A, iodinated protein A; sCD4, soluble CD4; sEGFR, soluble epidermal growth factor receptor; V domain, variable domain; VH domain, heavy chain variable domain. Back

2 C. Hanson, S. Karle, and S. Paul, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Robert Dannenbring and Yogesh Bangale for technical assistance. Peptide sequence assignments were by Dr. Richard Cook (Baylor College of Medicine Sequencing Facility). Soluble CD4 and HIV-1 isolates were obtained through the AIDS Research and Reference Reagent Program, NIAID, National Institutes of Health, with contributors Dr. Norbert Schuelke, Dr. Jay Levy, and the WHO-UNAIDS network.



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