Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.
Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.
Objective-Using intravenous injection of adeno-associated viral (AAV) vectors based on novel serotypes 7 and 8, we examined whether liver-specific expression of human apolipoprotein E (apoE) in apoE-deficient mice would completely prevent atherosclerosis after 1 year of sustained expression. Methods and Results-Chow-fed apoE Ϫ/Ϫ mice were injected via the tail vein with vectors based on AAV2 or novel serotypes AAV7 and AAV8 encoding human apoE3 driven by a liver-specific promoter. In contrast to the first-generation AAV2 vector, apoE levels of mice injected with chimeric AAV2/7 and AAV2/8 vectors reached Ϸ2-fold greater than normal human plasma levels by week 4 and maintained therapeutic levels up to 1 year. Cholesterol levels of AAV2/7-apoE and AAV2/8-apoE-treated mice were reduced to normal murine wild-type levels and were maintained for 1 year. At termination after 1 year, extensive atherosclerosis was present in the thoracic aortas and aortic roots of control AAV2/8-lacZ and AAV2-apoE-injected mice, but was completely prevented in both the AAV2/7 and AAV2/8-apoE-treated mice. Conclusion-We demonstrate that intravenous administration of AAV2/7-and AAV2/8-apoE vectors effectively mediated robust and sustained hepatic-specific expression of apoE and completely prevented atherosclerosis at 1 year.
We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL 2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL 2 , and HDL 3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL 2 .-Brown, R. J., G. C. Miller, N. Griffon, C. J. Long, and D. J. Rader. Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo. J. Lipid Res. 2007Res. . 48: 1132Res. -1139. Supplementary key words lipaseEndothelial lipase (EL) belongs to a superfamily of lipases (EC 3.1.1.3) that includes LPL and HL (1-6). These three lipases have both triglyceride (TG) lipase and phospholipase activity, but EL has relatively more phospholipase activity compared with LPL, which has predominantly TG lipase activity (7). Overexpression of EL in mice was shown to significantly reduce high density lipoprotein cholesterol (HDL-C) (1,8,9), whereas loss-of-function studies in mice result in significantly elevated plasma 10,11).In vitro and in vivo studies using chimeric proteins of LPL and HL have shown that the differences in substrate specificity between these two lipases are governed by a 22 amino acid loop, or "lid domain," within the N-terminal domain of the respective lipases that covers the catalytic site (12)(13)(14). The shorter 19 amino acid lid domain within EL partially contributes to its substrate specificity (15); other elements affecting substrate specificity remain to be elucidated.Human EL is translated as a 500 amino acid 57 kDa peptide that is processed into a mature 480 amino acid protein with an apparent molecular mass of 68 kDa after the loss of its signal peptide and the addition of N-linked glycosylation (1, 2). EL has five putative N-linked glycosylation sites [identified by the presence of asparagine-X-serine/threonine (Asn-Xaa-Ser/Thr) motifs]. We previously reported that four of the five sites, specifically are utilized (16). Abolishment of N-linke...
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