CHAPTER 4 Cloning, expression, and characterization of selected membrane proteins
4.4 RESULTS
4.4.9 Recombinant LIC13341 binds to the host extracellular matrix macromolecules
purified r-LIC13341 was validated for its secondary structure integrity before performing any biochemical analysis. The structural integrity of r-LIC13341 was evaluated by CD spectroscopy (Fig. 4.11A). Analysis of the experimental CD spectra data of r-LIC13341 by K2D2 program (Perez-Iratxeta and Andrade-Navarro, 2008) predicted 8.02% of α-helix and
22.14% of β-strand indicating its suitability for further biochemical studies. The secondary structure prediction of r-LIC13341 using the program PSIPRED (Buchan et al., 2013) was closer to the experimental CD data.
Several comprehensive studies on the adherence of L. interrogans to extracellular matrices of hosts suggested the existence of numerous adhesion molecules on the outer membrane of Leptospira which are surface exposed (Ghosh et al., 2018; Robbins et al., 2015). In such studies, Loa22, another outer membrane protein of Leptospira due to its moderate attachment to host ECM (Barbosa et al., 2006; Ghosh et al., 2018) is an ideal antigen for comparative analysis of antigen-ECM interaction. Herein, to investigate r-LIC13341 interaction with ECM ligands by ELISA, r-Loa22 along with its specific antibody was included in the study.
To evaluate LIC13341 interaction with the ECM components using ELISA, a soluble form of r-LIC13341 was allowed to bind to the immobilized host ECM components on microtiter plates. The antigen r-LIC13341 exhibited significant binding to laminin, fibronectin, collagen, hyaluronic acid, chondroitin sulfate A, elastin, and heparan sulfate compared to control protein fetuin or BSA (P<0.001) as a ligand (Fig. 4.11B). Among these extracellular matrices, laminin and hyaluronic acid showed the highest affinity for binding to r-LIC13341 on the microtiter plate. However, as anticipated, no specific binding of the antigen r-Loa22 to the target ECM macromolecules was detected in comparison to BSA or fetuin.
Nevertheless, the binding of r-LIC13341 with laminin and hyaluronic acid was also assessed on a quantitative basis (Fig. 4.11C). A dose-dependent and saturable binding were observed when increasing concentrations of the r-LIC13341 (0 - 24 µM) were allowed to bind to a fixed amount of laminin and hyaluronic acid (1 µg). The saturation level of r-LIC13341 binding to laminin and hyaluronic acid was reached at a concentration of 22 µM and 20 µM,
respectively (Fig. 4.11C). The calculated KD of the r-LIC13341 binding reactions was 420±57 nM and 299±35 nM to laminin and hyaluronic acid, respectively. In addition, the effect of the gradient increase in the amount of r-LIC13341 on the inhibition of live leptospires adherence to laminin and hyaluronic acid was examined by ELISA. The results indicate that the addition of r-LIC13341 in an increasing amount (0-10 µg) reduced the leptospiral adherence to both laminin and hyaluronic acid in a dose-dependent manner (Fig.
4.11D).
To examine the role of carbohydrate moieties of laminin in binding to r-LIC13341, laminin was oxidized by increasing concentrations of sodium metaperiodate (5 - 100 mM) at 4 C for 15 min. The inhibition of laminin binding to r-LIC13341 due to gradual increase in sodium metaperiodate concentration is shown in Fig. 4.12A. This mild treatment ensures cleavage of vicinal carbohydrate hydroxyl groups while the polypeptide chain structure remains intact (Woodward et al., 1985). The oxidation effect was dose-dependent and the r-LIC13341 attachment to metaperiodate-treated laminin got reduced to less than 50% at a 100 mM concentration of periodate (Fig. 4.12A). These results indicate that the carbohydrate residues of laminin are critical for the interaction of r-LIC13341 with this major ECM glycoprotein.
To further confirm the results of specific ECM binding, r-LIC13341 was heat-denatured before the binding reaction to hyaluronic acid. A sharp reduction in the binding of r- LIC13341 to hyaluronic acid under denaturing condition demonstrates the influence of antigen structure on the binding with hyaluronic acid (Fig. 4.12B). Moreover, the decline in binding under denaturing condition suggests conformational epitopes in LIC13341 are important for these interactions.
Figure 4.11. The recombinant-LIC13341 binds to host extracellular matrix components.
(A) Far-ultraviolet circular dichroism (CD) spectra of r-LIC13341. The spectra are depicted in the range of 190-260 nm wavelengths showing the presence of both α-helix and β-strand with a predominant signal of β-strand. CD spectra are shown as an average of 3 scans with a scanning speed of 100 nm min-1. (B) Enzyme-linked immunosorbent assay (ELISA) shows r-LIC13341 interaction with extracellular matrix components. Bovine serum albumin (BSA) and the highly glycosylated serum protein, fetuin, were used as controls for non-specific binding. Recombinant Loa22 was included as a negative control for non-specific binding with the ECM components.
Recombinant-LIC13341 exhibited significant binding to all ECM components compared to fetuin or BSA (P<0.001) and with a higher affinity towards laminin and hyaluronic acid. In contrast, Loa22 showed moderate binding with the ECM components used in this study. Results are indicative of two independent experiments. (C) Dose-dependent binding of the r-LIC13341 to laminin and hyaluronic acid. Laminin or hyaluronic acid coated microtiter plates were incubated with increasing concentrations of r-LIC13341 (0-24 µM). Binding of LIC13341 was measured using the anti-LIC13341 serum at an appropriate dilution. The mean absorbance values of r-LIC13341 binding to laminin and hyaluronic acid at 450 nm are shown. (D) Inhibition of Leptospira adherence to ECM in the presence of r-LIC13341 using ELISA. Laminin or hyaluronic acid (1 µg per well) was coated onto the microtiter plates followed by incubation with increasing concentrations of r-LIC13341 (0-10 µg) for 90 min at 37°C. Live L. interrogans serovar Copenhageni were added to microtiter plates and incubated for 90 min at 37°C. After washing, the quantification of bound leptospires was performed by anti-Loa22 serum. The
absorbance was measured at 450 nm wavelength using ELISA plate reader. The binding of L.
interrogans to laminin or hyaluronic acid in the absence of r-LIC13341 was considered as 100%
binding. A gradual reduction in Leptospira attachment to ECM was observed with increasing concentration of r-LIC13341. Error bars represent the standard deviations of the three replicates.
Figure 4.12. Binding of recombinant LIC13341 to laminin and hyaluronic acid is physio- chemically dependent. (A) Laminin oxidation reduces r-LIC13341-laminin interaction.
Immobilized laminin on microtiter plates was oxidized with various concentrations of sodium metaperiodate (5 to 100 mM) for 15 min at 4 C. The mean absorbance values of r-LIC13341 binding to oxidized or untreated laminin are compared. The reduction in the binding percentage of r-LIC13341 to oxidized laminin as a function of periodate concentration is indicated above each bar. (B) Heat-denatured r-LIC13341 fails to bind hyaluronic acid.
Hyaluronic acid coated (1 µg per well) microtiter plates were allowed to bind with increasing concentration of r-LIC13341 (0.25-4 µM) or its heat denatured form (∆r-LIC13341). Binding was detected using the anti-LIC13341 serum at an appropriate dilution. The mean absorbance values at 450 nm (± the standard errors of two independent experiments) are shown.
4.4.10 Recognition of recombinant LIC13341 by serum antibodies of human and