Role of UDP-N-acetylglucosamine (GlcNAc) and O-GlcNAcylation of hyaluronan synthase 2 in the control of chondroitin sulfate and hyaluronan synthesis.
Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t(&frac12;) >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies.
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Davide
D
Vigetti
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Sara
S
Deleonibus
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Paola
P
Moretto
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Eugenia
E
Karousou
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Manuela
M
Viola
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Barbara
B
Bartolini
Biomedical Engineering ND20, The Cleveland Clinic, Cleveland, Ohio 44195.
Vincent C
VC
Hascall
Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland.
Markku
M
Tammi
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy.
Giancarlo
G
De Luca
Dipartimento di Scienze Chirurgiche e Morfologiche, Università degli Studi dell'Insubria, via J. H. Dunant 5, 21100 Varese, Italy. Electronic address: alberto.passi@uninsubria.it.
Alberto
A
Passi
D019943:Amino Acid Substitution; D001011:Aorta / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000096:biosynthesis* / Q000235:genetics; D003925:Diabetic Angiopathies / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D020125:Mutation, Missense; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000235:genetics / Q000378:metabolism*
D009124:Muscle Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases