Berryman D E, Bensadoun A
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1993 Feb 15;268(5):3272-6.
Lipoprotein lipase (LPL) binds to heparin and heparan sulfate proteoglycans. We have employed site-directed mutagenesis to dissect one of the proposed heparin binding domains of avian LPL, which contains the sequence Arg-Lys-Asn-Arg (amino acids 281-284). Various single, double, and triple mutants of chicken LPL were constructed in order to alter the positive charge of this region. The mutant and wild-type cDNAs were subcloned into an expression vector, pRc/CMV, and expressed in Chinese hamster ovary cells. In general, the LPL mutants with a decrease in regional positive charge showed a decrease in affinity for heparin and heparan sulfate proteoglycans. The greatest effect was seen with the triple mutant, LPL 5G, in which all of the positively charged amino acids were altered to neutral residues. On a heparin-Sepharose column, LPL 5G eluted at 0.96 M NaCl compared with 1.35 M for wild-type LPL. This mutant also had the lowest specific activity with 1.5 mu eq fatty acid/micrograms/h for the cell-associated pool and with no detectable activity in the media. Wild-type cells, however, produced a lipase with a specific activity of 12.4 and 13.1 mu eq fatty acid/micrograms/h for cell-associated and media lipase pools, respectively. LPL 5G also showed a decrease in affinity for the heparan sulfate proteoglycans on the cell surface of Chinese hamster ovary cells. In conclusion, the region of avian LPL between Arg281 and Arg284 does appear to be involved in heparin-binding; however, additional regions must be involved since binding was not completely abolished. In addition, specific activity of the cell-associated and secreted LPL is correlated to affinity of the enzyme for heparan sulfate chains.
脂蛋白脂肪酶（LPL）可与肝素及硫酸乙酰肝素蛋白聚糖结合。我们运用定点诱变技术剖析了禽源LPL的一个假定的肝素结合结构域，该结构域包含序列精氨酸-赖氨酸-天冬酰胺-精氨酸（氨基酸281 - 284）。构建了鸡LPL的各种单突变、双突变和三突变体，以改变该区域的正电荷。将突变型和野生型cDNA亚克隆到表达载体pRc/CMV中，并在中国仓鼠卵巢细胞中表达。一般来说，区域正电荷减少的LPL突变体对肝素和硫酸乙酰肝素蛋白聚糖的亲和力降低。三突变体LPL 5G的效果最为显著，其中所有带正电荷的氨基酸都被改变为中性残基。在肝素 - 琼脂糖柱上，LPL 5G在0.96 M NaCl浓度下洗脱，而野生型LPL在1.35 M NaCl浓度下洗脱。该突变体的比活性也最低，细胞相关池的比活性为1.5 μeq脂肪酸/微克/小时，培养基中未检测到活性。然而，野生型细胞产生的脂肪酶，细胞相关和培养基脂肪酶池的比活性分别为12.4和13.1 μeq脂肪酸/微克/小时。LPL 5G对中国仓鼠卵巢细胞表面的硫酸乙酰肝素蛋白聚糖的亲和力也降低。总之，禽源LPL中精氨酸281和精氨酸284之间的区域似乎确实参与肝素结合；然而，由于结合并未完全消除，必然还涉及其他区域。此外，细胞相关和分泌型LPL的比活性与该酶对硫酸乙酰肝素链的亲和力相关。
