The pH-dependence of pepsin-catalysed reactions

Cornish‐Bowden, Athel; Knowles, Jeremy R.

doi:10.1042/bj1130353

Cited by 96 publications

(53 citation statements)

References 27 publications

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“…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 M did not cleave Bt-gp120 detectably (not shown; reaction conditions as in Fig.…”

Section: Catalytic Activity Of Polyclonal Igm Abs-eachsupporting

confidence: 71%

Naturally Occurring Proteolytic Antibodies

Paul

Karle

Planque

et al. 2004

Journal of Biological Chemistry

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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 IgMcatalyzed 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 Lys 432 -Ala 433 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 Lys 432 -Ala 433 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.As the first class of Abs 1 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 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...

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Section: Catalytic Activity Of Polyclonal Igm Abs-eachsupporting

confidence: 71%

Naturally Occurring Proteolytic Antibodies

Paul

Karle

Planque

et al. 2004

Journal of Biological Chemistry

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“…Hapten CRA I was validated previously as a probe for nucleophilic reactivities expressed by serine proteases, including IgG Abs (11,28). The extent of irreversible CRA binding activity correlates approximately with the catalytic activity (11,29).…”

Section: Discussionmentioning

confidence: 99%

“…Next, we considered the possibility that pepsin released from the column could be responsible for the observed Fab activity. The pH optimum of pepsin is 1.5-2.7 depending on the substrate (28). The catalysis assays were repeated in 0.1 M glycine, pH 2.7, 1 mM CHAPS.…”

Section: Irreversible Cra-b Cell Binding-haptenmentioning

confidence: 99%

Ontogeny of Proteolytic Immunity

Planque

Bangale

Song

et al. 2004

Journal of Biological Chemistry

104

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Antigen-specific IgG Abs 1 in autoimmune and alloimmune disease are described to catalyze chemical reactions (1-3). Examples of catalytic Abs raised by routine experimental immunization with ordinary antigens have also been published (4 -7). However, no consensus has developed whether naturally occurring catalytic Abs represent rare accidents arising from adaptive sequence diversification processes or genuine enzymes with important functional roles. The major reason is that the turnover (k cat ) of antigen-specific IgG Abs is low. Some catalytic Abs express catalytic efficiencies (k cat /K m ) comparable to conventional enzymes, but this is due to high affinity recognition of the antigen ground state (reviewed in Ref. 8).Certain enzymes cleave peptide bonds by a mechanism involving the formation of a transient covalent intermediate of the substrate and a nucleophilic residue present in the active site. The nucleophiles are generated by intramolecular activation mechanisms, e.g. the activation of Ser/Thr side chain hydroxyl groups by hydrogen bonding to His residues, and can be detected by covalent binding to electrophilic phosphonate diesters (9, 10). Using these compounds as covalently reactive analogs of antigens (CRAs), we observed that IgG Abs express nucleophilic reactivities comparable to trypsin (11). Despite their nucleophilic competence, IgG Abs display low efficiency proteolysis, presumably due to deficiencies in steps occurring after formation of the acyl-Ab intermediate, viz., water attack on the intermediate and product release. Occupancy of the B cell receptor (BCR, surface Ig complexed to ␣ and ␤ subunits along with other signal transducing proteins) by the antigen drives B cell clonal selection. Proteolysis by the BCR is compatible with clonal selection, therefore, only to the extent that the release of antigen fragments is slower than the rate of antigeninduced transmembrane signaling necessary for induction of cell division. Immunization with haptens mimicking the charge characteristics of the transition state (12) has been suggested as a way to surmount the barrier to adaptive improvement of catalytic rate constants. Catalysis by designer IgG Abs obtained by these means, however, also proceeds only slowly.In mice and humans, the initial Ab repertoire consists of ϳ100 heritable VL and VH genes. Adaptive maturational processes expand the repertoire by several orders of magnitude. The initial BCR complex on the pre-B cell surface contains V-(D)-J rearranged Ig chains as a complex with surrogate L chains (reviewed in Ref. 13). Precise assignment of the B cell differentiation stage at which cell division becomes antigen-dependent is somewhat ambiguous, but it is generally believed that non-covalent antigen binding to the pre-BCR is not required for initial cell growth. / chains replace the surrogate L chain at the later stages of antigen-driven B cell differentiation, which is accompanied by diversification via somatic hypermutation processes and continued gene rearrangements (14,15). V-(D)-J gene ...

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“…Newborns and young infants secrete both acid and pepsin during fasting, but do not respond to the usual stimuli with increased acid or pepsin output (1, 7). The lowest intragastric pH and highest concentration of pepsin would then occur just before a meal; ingestion of breast milk rapidly neutralizes the acid, dilutes, and inactivates the pepsin (20,27,5). The fall in pH after a breast milk meal probably occurs much more slowly than does emptying of the stomach; therefore, in vitro conditions used in this study are probably more severe than occurs in most infants.…”

Section: Discussionmentioning

confidence: 96%