Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity

Kirsi Rilla,Hanna Siiskonen,Andrew P Spicer,Juha M.T Hyttinen,Markku I Tammi,Raija H Tammi

doi:10.1074/jbc.m504736200

Kirsi Rilla, Hanna Siiskonen + Show 4 more

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https://doi.org/10.1074/jbc.m504736200

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Abstract

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

Highlights

Hyaluronan is a high molecular mass, unbranched acidic glycosaminoglycan found in the extracellular matrix of many vertebrate tissues
To show that the transfected green fluorescent protein (GFP)-Hyaluronan synthases (HASs) were functional in the rat epidermal keratinocyte cell line (REK) cells, the amount of hyaluronan released into growth medium was measured using an ELSA assay (Fig. 1)
The content of hyaluronan in the medium was clearly higher in the cultures transfected with GFP-Has2 and GFP-Has3 constructs than in those transfected with the empty GFP vector (Fig. 1)

Summary

Introduction

Hyaluronan is a high molecular mass, unbranched acidic glycosaminoglycan found in the extracellular matrix of many vertebrate tissues. Hyaluronan synthases (HASs) are unique enzymes that synthesize hyaluronan from UDP-activated N-acetylglucosamine and glucuronic acid and coordinately translocate the growing chain through the plasma membrane [2]. Hyaluronan synthases are integral membrane proteins with an active site located in the cytoplasmic side of the plasma membrane [2] In this respect, hyaluronan differs from other complex carbohydrates, the synthesis of which begins in the endoplasmic reticulum and is completed in the Golgi apparatus. Expression of enzymatically active HASs as GFP-tagged fusion proteins provides an opportunity to 1) localize the enzymes directly in living cells, 2) follow their movements subcellularly, 3) examine possible regulatory points in their ability to synthesize hyaluronan, and 4) detect the influence of elevated hyaluronan synthesis rate on important cellular functions, such as those described previously in keratinocytes [15]

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