Cloning, Sequencing and Functional Overexpression of the Streptococcus equisimilis H46A gapC Gene Encoding a Glyceraldehyde‐3‐phosphate Dehydrogenase that also Functions as a Plasmin(ogen)‐Binding Protein

Abstract

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase (GraP-DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin (ogen) To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichiu cnli from QIAexpress expression plasmids to yield the authentic GapC or (His),GapC carrying a hexahistidyl N-terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP-DH activities of about 80 kat/mol ( 4 3 0 Uimg) after purification. Their binding parameters [association (k,,) and dissociation (kc,) rate constants, and equilibrium dissociation constants (KCI = k&J] for the interaction with human Gluplasminogen and plasmin were determined by real-time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP-DH from Bacillus steurothermuphilus (RstGraP-DH), a nonpathogenic organism, was included in these experiments. The K,, values for binding of plasininogen to GapC, (His),GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme posse low-affinity binding sites for the gupC gene product and that the hexahistidyl terminus does not affect function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-terminal lysine residues for binding to the zymogen.Keywcwds: Streptococcus equisimilis; glyceraldehyde-3-phosphate dehydrogenase; gapC gene; plasmin-(ogen)-binding protein.As a major surprise of recent investigations into the nature of cell surface proteins of the pathogenic streptococci, a wallassociated plasmin binding protein has been discovered that structurally and functionally belongs to the glyceraldehyde-3- Ahhrc.iYntions. BHI, brain heart infusion ; GraP-DH, generic abbreviation [or glyceraldehyde-3-phosphate dehydrogenases: BstGraP-DH, GraP-DH from B~d r ' i i s .stearotlirrmr~philu.~ ; IPTG, isopropyl p-u-thiogalactopyranoside; LBS, lysine-binding site; Ni-NTA, Ni2--nitrilotriacetic acid; ~-~~l-Leii-Lys-CH,Cl, i~-valyl-leucyl-lysyI-chloromethanethane.En~rtzes. Glycelaldel?yde-3-phosphate dehydrogenase (EC 1.2.1.12); plasmin (EC 3.4.21.7).Note. The novel nucleotide sequence data published here has been deposited with the EMBLIGenBanWDDBJ sequence data banks and is available under accesibion number X97788. Broder et al. (1991) were the first to purify from the surface of a group A stre...

Identification of Major Outer Surface Proteins of Streptococcus agalactiae

“…Also in Schistosoma, a specific epitope of the enzyme glyceraldehyde-3-phosphate dehydrogenase has been identified as a vaccine candidate (1). Interestingly, the same protein has been identified as a plasmin(ogen) binding protein on Streptococcus equisimilis (12), the same function assigned to surface-located enolase in Streptococcus pyogenes (23,30). Demonstrating the multiple functions that the same protein may possess, glyceraldehyde-3-phosphate dehydrogenase has also been identified as a transferrin binding protein (24).…”

Identification, Recombinant Expression, Immunolocalization in Macrophages, and T-Cell Responsiveness of the Major Extracellular Proteins of Francisella tularensis

“…It is the major transferrin-binding protein in the cell walls of staphylococci (23). It is also identified as a major surface protein in group A streptococci that is capable of binding fibronectin, plasminogen, lysozyme, and actin (11,27). In addition, glyceraldehyde-3-phosphate dehydrogenase and DnaK of Listeria monocytogenes are present in the cell wall and show strong binding affinity to plasminogen (33).…”

Spatial Regulation and Activity Modulation of Plasmin by High Affinity Binding to the G domain of the α3 Subunit of Laminin-5

“…To test this hypothesis, the affinity of Pg and tPA binding to laminin-5 was quantified by surface plasmon resonance in an evanescent wave biosensor. Purified laminin-5 was added stepwise in increasing concentrations (0 -150 nM) to immobilized Pg or tPA, and the degree of refraction of a light beam crossing the surface of the immobilized protein was used to measure the affinity of protein-protein interaction (20,21). Laminin-5 binds to immobilized Pg and tPA in a concentration-dependent, saturable fashion with dissociation constants (K d ) of 40 and 35 nM, respectively (Fig.…”