Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

Abstract

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant.

Expression and Characterization of a Soluble, Active Form of the Jaagsiekte Sheep Retrovirus Receptor, Hyal2

“…In our case, the extreme stability of the complex suggests that its formation is essentially irreversible, particularly considering the additional effects of avidity and rebinding due to the higher local concentrations of two membrane-anchored interactors. Such slow dissociation kinet- (22). The high affinity of JSU-IgG for sHyal2 and the resultant long half-life of the complex indicates that JSRV Env is highly proficient in its ability to anchor incoming virus particles onto target cells expressing human Hyal2, and presumably onto cells expressing sheep Hyal2 as well.…”

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

“…His-tagged rSQAPI was eluted from the column with 20 mM sodium phosphate buffer, pH 6.0, containing 500 mM NaCl and 50 mM EDTA. Fractions containing His-tagged rSQAPI were pooled and concentrated, and the buffer was exchanged into 10 mM KH 2 PO 4 , pH 3.0, containing 0.2% NaN 3 .…”

“…A novel proteinaceous aspartic acid proteinase inhibitor (SQAPI) has been characterized from squash phloem exudate (Cucurbita maxima Duchesne) (2,3). The protein is distinct from the other six families of proteinaceous aspartic PIs that have been identified and had their genes cloned: the potato plant Kunitz inhibitors (4), the Ascaris inhibitors (5), the yeast proteinase A inhibitor-3 (6), a domain of the sea anemone thyroglobulin type-1 inhibitor (7,8), the pig serpin inhibitor (9), and the Bacillus sp.…”

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis

“…pepstatine, to show that erythrocyte cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis and degradation of the membrane proteins [32]. Some additional examples of aspartic proteinase inhibitors are acetyl-pepstatine, an inhibitor of HIV proteinase [33], recombinant squash aspartic peptidase inhibitor (rSQAPI), a reversible inhibitor of aspartic proteinases [34], HLTV-1 retroviral protease inhibitors [35], human immunodeficiency virus (HIV)-1 diaminodiol reversible inhibitors [26], a 2 -macroglobulin [36,37].…”