The Sulfate Groups of Chondroitin Sulfate- and Heparan Sulfate-containing Proteoglycans in Neutrophil Plasma Membranes Are Novel Binding Sites for Human Leukocyte Elastase and Cathepsin G

Edward J Campbell,Caroline A Owen

doi:10.1074/jbc.m608346200

Edward J Campbell, Caroline A Owen

Open Access

https://doi.org/10.1074/jbc.m608346200

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Abstract

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. (125)I-Labeled HLE and CG bind to PMN at 0 degrees C in a saturable and reversible manner (K(D) = 5.38 and 4.36 x 10(-7) m and 11.5 and 8.1 x 10(6) binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.

Highlights

Until recently little has been known about the mechanisms by which HLE and CG mediate their activities in vivo because 1) minimal amounts of these enzymes are freely released from viable PMN activated with biologic mediators [9, 10], 2) CG is poorly soluble in isotonic solutions [11], and 3) plasma and interstitial fluids contain abundant, high affinity inhibitors of serine proteinases
We report that the sulfate groups of chondroitin sulfate- and heparin sulfate-containing proteoglycans (CSPG and HSPG) in PMN plasma membranes are high volume, low affinity binding sites for HLE and CG, which are well suited to bind the millimolar concentrations of serine proteinases generated at the cell surface during PMN degranulation
The binding data were corrected for non-specific binding of ligands and analyzed by the Scatchard method to determine the number of binding sites per cell and the binding affinity (KD) of the PMN plasma membrane sites for HLE and CG on PMN. b Data are the mean Ϯ S.E.; n ϭ 4 separate experiments performed in quadruplicate

Summary

Introduction

Until recently little has been known about the mechanisms by which HLE and CG mediate their activities in vivo because 1) minimal amounts of these enzymes are freely released from viable PMN activated with biologic mediators [9, 10], 2) CG is poorly soluble in isotonic solutions [11], and 3) plasma and interstitial fluids contain abundant, high affinity inhibitors of serine proteinases. We report that the sulfate groups of chondroitin sulfate- and heparin sulfate-containing proteoglycans (CSPG and HSPG) in PMN plasma membranes are high volume, low affinity binding sites for HLE and CG, which are well suited to bind the millimolar concentrations of serine proteinases generated at the cell surface during PMN degranulation. Cell surface binding of HLE and CG arms PMN with locally active HLE and CG, which contribute important activities in health and in disease processes

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Discussion

Conclusion