A Novel Carbohydrate-Glycosphingolipid Interaction between a β-(1–3)-Glucan Immunomodulator, PGG-glucan, and Lactosylceramide of Human Leukocytes

Janet W Zimmerman,Johanna Lindermuth,Pamela A Fish,Gerard P Palace,Tom T Stevenson,Duane E Demong

doi:10.1074/jbc.273.34.22014

Abstract

The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.

Highlights

Crude preparations of yeast ␤-glucans have been known for over 40 years to stimulate animal defense mechanisms, and it is generally believed that the active component in these preparations is ␤-(1–3)-glucans. ␤-(1–3)-Glucans are major components of yeast and fungal cell walls, this stimulation may reflect a natural defense in response to breakdown products of the fungal cell wall
This glycolipid is especially abundant in human neutrophils where it makes up two-thirds of the total glycolipids, with approximately 20% found in the cell membrane [20, 21]
Binding Activity from Human Leukocyte Membranes Copurifies with LacCer—Extraction of human leukocyte membranes with chloroform/methanol results in two phases and a proteinaceous interphase

Summary

Introduction

Crude preparations of yeast ␤-glucans have been known for over 40 years to stimulate animal defense mechanisms, and it is generally believed that the active component in these preparations is ␤-(1–3)-glucans. ␤-(1–3)-Glucans are major components of yeast and fungal cell walls, this stimulation may reflect a natural defense in response to breakdown products of the fungal cell wall. ␤-(1–3)-Glucans are major components of yeast and fungal cell walls, this stimulation may reflect a natural defense in response to breakdown products of the fungal cell wall Both soluble and particulate ␤-glucans have numerous biological activities including stimulation of the reticuloendothelial system, induction of hematopoiesis, activation of the complement and/or cytokine system, inhibition of tumor cell growth, and induced resistance to infections (for reviews, see Ref. 1 and 2). In efforts to characterize the receptor responsible for these functions, it was shown that binding of radiolabeled PGG-glucan could be detected in membranes from human leukocytes, and that the binding was due primarily to the neutrophil content.

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