Packaging of Proteases and Proteoglycans in the Granules of Mast Cells and Other Hematopoietic Cells

doi:10.1074/jbc.270.33.19524

Packaging of Proteases and Proteoglycans in the Granules of Mast Cells and Other Hematopoietic Cells

A CLUSTER OF HISTIDINES ON MOUSE MAST CELL PROTEASE 7 REGULATES ITS BINDING TO HEPARIN SERGLYCIN PROTEOGLYCANS (*)

From the (1)Department of Medicine, Harvard Medical School and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the
(2) Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138

^¶ To whom all correspondence should be addressed:
Dept. of Medicine, Harvard Medical School, Seeley G. Mudd Bldg., 250 Longwood Ave., Boston, MA 02115.
Tel: 617-432-2838; Fax: 617-432-0979; rstevens{at}warren.med.harvard.edu

Abstract

Mouse mast cell protease 7 (mMCP-7) is a tryptase stored in the secretory granules of mast cells. At the granule pH of 5.5, mMCP-7 is fully active and is bound to heparin-containing serglycin proteoglycans. To understand the interaction of mMCP-7 with heparin inside and outside the mast cell, this tryptase was first studied by comparative protein modeling. The “pro” form of mMCP-7 was then expressed in insect cells and studied by site-directed mutagenesis. Although mMCP-7 lacks known linear sequences of amino acids that interact with heparin, the three-dimensional model of mMCP-7 revealed an area on the surface of the folded protein away from the substrate-binding site that exhibits a strong positive electrostatic potential at the acidic pH of the granule. In agreement with this calculation, recombinant pro-mMCP-7 bound to a heparin-affinity column at pH 5.5 and readily dissociated from the column at pH > 6.5. Site-directed mutagenesis confirmed the prediction that the conversion of His residues 8, 68, and 70 in the positively charged region into Glu prevents the binding of pro-mMCP-7 to heparin. Because the binding requires positively charged His residues, native mMCP-7 is able to dissociate from the protease/proteoglycan macromolecular complex when the complex is exocytosed from bone marrow-derived mast cells into a neutral pH environment. Many hematopoietic effector cells store positively charged proteins in granules that contain serglycin proteoglycans. The heparin/mMCP-7 interaction, which depends on the tertiary structure of the tryptase, may be representative of a general control mechanism by which hematopoietic cells maximize storage of properly folded, enzymatically active proteins in their granules.

Footnotes

↵^§ Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. Current address: The Rockefeller University, 1230 York Ave., New York, NY 10021.
↵* This work was supported by Grants AI-23483, AR-07530, AR-36308, GM-30804, and HL-36110 from the National Institutes of Health, by a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research, and by a grant from the Jane Coffin Childs Memorial Fund for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 The abbreviations used are:

mMC-CPA

mouse mast cell carboxypeptidase A

mBMMC

mouse bone marrow-derived mast cells

mMCP

mouse mast cell protease

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

CAPS

3-[cyclohexylamino]-1-propanesulfonic acid

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
↵²MODELLER is available by anonymous FTP from tammy.harvard. edu:pub/modeler and also as part of QUANTA (MSI, Burlington, MA; jcollins{at}msi.com)
↵³A. D. MacKerell, Jr., D. Bashford, M. Bellott, R. L. Dunbrack, Jr., M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D. Joseph, L. Kuchnir, K. Kuczera, F. T. K. Lau, C. Mattos, S. Michnick, T. Ngo, D. T. Nguyen, B. Prodhom, B. Roux, M. Schlenkrich, J. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiorkiewicz-Kuczera, and M. Karplus, manuscript in preparation.
↵⁴N. Ghildyal, M. F. Gurish, D. S. Friend, K. F. Austen, and R. L. Stevens, unpublished data.
- Received March 13, 1995.
- Revision received May 9, 1995.