title: NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia: NEU1 restrains endothelial cell migration, whereas NEU3 does not.
abstract: The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.
authors.affiliation: Center for Vaccine Development, University of Maryland, School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Cross
authors.forename: Sang Won
authors.initials: SW
authors.lastname: Hyun
authors.forename: Alba
authors.initials: A
authors.lastname: Miranda-Ribera
authors.forename: Chiguang
authors.initials: C
authors.lastname: Feng
authors.forename: Anguo
authors.initials: A
authors.lastname: Liu
authors.forename: Chinh
authors.initials: C
authors.lastname: Nguyen
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Irina G
authors.initials: IG
authors.lastname: Luzina
authors.forename: Sergei P
authors.initials: SP
authors.lastname: Atamas
authors.forename: William S
authors.initials: WS
authors.lastname: Twaddell
authors.forename: Wei
authors.initials: W
authors.lastname: Guang
authors.forename: Erik P
authors.initials: EP
authors.lastname: Lillehoj
authors.forename: Adam C
authors.initials: AC
authors.lastname: Puché
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
authors.forename: Antonino
authors.initials: A
authors.lastname: Passaniti
authors.forename: Simeon E
authors.initials: SE
authors.lastname: Goldblum
mesh_terms: D001011:Aorta / Q000201:enzymology; D002339:Carotid Arteries / Q000201:enzymology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002465:Cell Movement; D002467:Cell Nucleus / Q000201:enzymology; D002536:Cerebral Arteries / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000494:pharmacology; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D018613:Microscopy, Confocal; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
chemical_list: C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; C486385:NEU1 protein, human; C403285:NEU2 protein, human; C491428:NEU4 protein, human; C465436:Neu3 protein, human; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid