NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia

• Published in: The Journal of biological chemistry 2012-05, Vol.287 (19), p.15966-15980
• Main Authors: Cross, Alan S., Hyun, Sang Won, Miranda-Ribera, Alba, Feng, Chiguang, Liu, Anguo, Nguyen, Chinh, Zhang, Lei, Luzina, Irina G., Atamas, Sergei P., Twaddell, William S., Guang, Wei, Lillehoj, Erik P., Puché, Adam C., Huang, Wei, Wang, Lai-Xi, Passaniti, Antonino, Goldblum, Simeon E.
• Format: Article
• Language: English
• Subjects: Endothelial Dysfunction; Glycobiology; Lung; Migration; Neuraminidase; Sialic Acid; Sialidase; Signal Transduction; Vascular Biology; Wound Healing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.346817

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. Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia express catalytically active NEU1 and NEU3 sialidases, and NEU1 restrains the endothelial migratory response to wounding. Conclusion: NEU1 regulates endothelial remodeling in response to injury. Significance: Learning how NEU1 and NEU3 regulate sialylated molecules on the endothelial surface is key to understanding endothelial receptor-ligand, cell-cell, and host-pathogen interactions.