Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Shao-Chen Lee,Hong-Hsiang Guan,Chia-Hui Wang,Wei-Ning Huang,Siu-Cin Tjong,Chun-Jung Chen,Wen-Guey Wu

doi:10.1074/jbc.m412398200

Abstract

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

Highlights

Citrate is present as a major component of snake, bee, scorpion, and ant venom and serves as counter ion for the basic polypeptides of many venoms [1]
There was no significant difference of the cell retention of CTX A3 by mutant cells and wild-type cells (CHO-K1 expressing GAGs) in Hanks’ balanced salt solution (HBSS), the addition of 10 mM citrate significantly increased toxin retention by wild-type cells but not mutant cells. These results suggest that GAGs on CHO cell surfaces play a role in mediating the retention of CTX A3 in the presence of citrate
We developed a new method for studying the cell retention behavior of animal toxins, and we showed that citrate played a role in the heparan sulfate (HS)-mediated cell retention of CTX A3

Summary

EXPERIMENTAL PROCEDURES

Materials—Crude snake venom (N. atra), heparin, and other chemicals were purchased from Sigma. Anti-cardiotoxin antibody was prepared by immunoprecipitation of horse antiserum (Center for Disease Control Taiwan, Taipei, Taiwan) with CTXs. Cell Retention Tests—Chinese hamster ovary cells (CHO-K1, ATCC number CCL-61) and GAG biosynthesis deficiency mutant CHO cells (pgsA745, ATCC number CRL-2242) were maintained in modified McCoy’s 5A medium (Sigma) supplemented with 10% (v/v) fetal calf serum (Hyclone), 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Cells in the microcapillary tube were washed with Hanks’ balanced salt solution (HBSS) before CTX A3 application. Control experiments with mouse anti-␤actin antibody and anti-mouse-IgG antibody conjugated to horseradish peroxidase were performed to ensure that the cells were evenly immobilized. CTX A3 retention by the cells was detected by immunostaining with a horse anti-CTX-antibody (1:2000) and a secondary anti-horse IgG antibody (1:2000) conjugated with horseradish peroxidase (Abcam, Inc., Cambridge, MA). After washing away unbound antibody, a chemiluminescence detection reagent (ECL, Amersham Biosciences) was added

TABLE I

No protein molecules per asymmetric unit

RESULTS

TABLE III Comparison in primary sequences of CTX homologues

DISCUSSION