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

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.

Hyaluronan-dependent pericellular matrix☆

“…Disruption of the cell coat with oligosaccharides prevented cell spreading, and is at least consistent with this idea [15]. Studies examining localization of GFP-tagged HAS indicate that the initial adhesion during extension of a filopodium may be mediated by the hyaluronan that is emanating from the synthase located at the tip and along the sides of the filopodium [30]. This could promote filopodial growth in that direction.…”

“…This occurs even though the cells do not express significant amounts of aggregating proteoglycans. The coat formation is even more pronounced in epithelial cells transfected with GFP-Has2 and GFP-Has3 [30,31] (Fig. 4).…”

“…Promotion of lammellipodial formation was initiated by application of exogenous hyaluronan to an "inactive" cell edge with a micropipette, suggesting that hyaluronan may promote the formation of cell protrusions and has a chemotactic effect [102]. Another study showed that the enzymatic activity of HAS is coupled to plasma membrane residence and the HAS is localized to cell protrusions [30]. In this recent study, HAS overexpression and hyaluronan production was also found to influence the formation of these microvillous protrusions in adenocarcinoma cells and other epithelial cells [31] (Fig.…”

“…For example, hyaluronan cable structures that arise from within a perinuclear location and connect multiple cell nuclei over long distances form the basis for a novel, mechanically continuous network between cells [47]. Other data suggest that there is a pool of latent HAS in these perinuclear locations that can be activated by various stimuli [30,38]. We have previously observed intracellular hyaluronan in close proximity to nuclear clefts and furrows, as well as binding of exogenous hyaluronan to nucleoli [88].…”

Hyaluronan Synthesis Induces Microvillus-like Cell Surface Protrusions

“…There are distant homologues of the vertebrate HAS also in Streptococcus and Pasteurella bacteria (24). All vertebrate HAS isoenzymes are apparently synthesized into the membrane of the endoplasmic reticulum and transported through Golgi apparatus to the plasma membrane (25,26). To be enzymatically active, HASs are not known to bind any other protein (27).…”