The Interaction Between Lipoprotein(a) And Recombinant Aspartase Derived From Nontypeable Haemophilus Influenzae

Nontypeable Haemophilus influenzae (NTHi) can adhere to the host ECM components, like laminin, fibronectin and various collagens. Meanwhile, NTHi can aslo bind plasminogen (Pig) via its Pig receptors. Pig recruited on NTHi cell can be activated to plasmin (Pm) by tissue-type plasminogen activator, resulting in the formation of bacterium-bound proteolytic Pm activity and the degradation of host tissue barriers, contributing to the effective dissemination and invasion of NTHi. Lipoprotein(a) consists of apolipoprotein(a) [Apo(a)] and low-density lipoprotein (LDL). Apo(a) shares a high degree of homology with the human plasminogen (Pig), and both of them contain lysine-binding sites (LBS). Taken together, Lp(a) may competitively bind aspartase on the NTHi surface and then inhibit the binding of Pig to NTHi.In present research, the interaction of human plasma Lp(a) with recombinant nontypeable Haemophilus influenzae aspartase was studied.Recombinant aspartase (rASP), and C-terminal lysine-deleted variant of ASP (rASP△K) were cloned and expressed in E.coli BL21 (DE3). Binding of recombinant proteins to Lp(a) was demonstrated with enzyme-linked immunosorbent assay (ELISA), binding-inhibition ELISA, affinity chromatography-binding assay and Western blot analysis. The results showed that rASP bound to Lp(a) specifically, but rASP△K did not, indicating that C-terminal lysine residue of rASP was the only binding site. Menwhile, rASP couldn’t interacted with LDL, and the inhibition of EACA to the binding of rASP and Lp(a) was dose-dependent (1 mM EACA suppressed the binding of Lp(a) to rASP completely), revealing that the linkage between Lp(a) and rASP was mediated by lysine residue of rASP and the LBS of Apo(a). In addition, the association of Pig with rASP was reduced by 14.7% and 30.3%, in which Lp(a) was used at 50 ng and 100 ng, indicating that Lp(a) could inhibit the binding of Pig to rASP, which was dose-dependentIt was demonstrated for the first time that Lp(a) via its LBS in Apo(a) bound to the C-terminal lysine of rASP, and Lp(a) could inhibit the binding of Pig to rASP.