Functional characterization of protease-treated Bacillus anthracis protective antigen.

J.M Novak,M.P Stein,S.F Little,S.H Leppla,A.M Friedlander

doi:10.1016/s0021-9258(18)41911-4

Abstract

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.

Highlights

Characterization of the functional domains of BaciZZus anthracis protective antigen (PA, 83-kDa),the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is importantfor understanding the mechanism of entry and action of the anthrax toxins
We found whereas cleavage of P A at the trypsinsite was absolutely required for generation of a biologically active molecule, chymotrypsin treatment resulted in PA preparations that were factor; EF, edema factor; 83-kDa PA, non-cleaved PA; T- biologically inactive
PA is essential for cell binding and internalization of both anthrax edema toxin and anthrax lethal toxin [1, 3]

Summary

THEJOURNAL OF BIOLOGICCAHLEMISTRY

Vol 267, No 24, Issue of August 25, pp. 17186-17193.1992 Printed in U.S.A. Functional Characterizationof Protease-treated Bacillus anthracis Protective Antigen*. Treatment with both chymotrypsin and trypsin generated threemajor fragments, 20, “17,”and 47 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis This P A preparation was biologically inactive. We found whereas cleavage of P A at the trypsinsite was absolutely required for generation of a biologically active molecule, chymotrypsin treatment resulted in PA preparations that were factor; EF, edema factor; 83-kDa PA, non-cleaved (native) PA; T- biologically inactive. PA, trypsin-treatedPA; Ch-PA, chymotrypsin-treated PA; Ch/T-PA, treated preparation of PA by sequentially treating with chychymotrypsin/trypsin-treated Hank’s balanced salt solution; PA; PBS, BSAb,ovine serumalbumin; phosphate-buffered saline; SDS, motrypsin, followed by trypsin This dual-protease treatment sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; resulted in a preparation containing a 47-, a “17,”- and a 20-. In thisstudy we investigated whether thbeiologically inactive chymotrypsin-treated PA preparationswere defective for bindingto the cellular receptor or binding and internalizationof lZ5I-LF

MATERIALS ANDMETHODS

RESULTS

GLU l pin Chymotrypsin ot’

DISCUSSION