De Angelis D A, Braun P E
Biochemistry Department, McGill University, Montreal, Quebec, Canada.
J Neurochem. 1996 Jun;66(6):2523-31. doi: 10.1046/j.1471-4159.1996.66062523.x.
The binding of 2', 3'-cyclic nucleotide 3'-phosphodiesterase isoform 1 (CNP1) to myelin and its association with cytoskeletal elements of the sheath have been characterized with in vitro synthesized polypeptides and purified myelin. We have previously shown that the cysteine residue present in the carboxy-terminal CXXX box of CNP1 is isoprenylated, and that both C15 farnesyl and C20 geranylgeranyl isoprenoids can serve as substrates for the modification. Here, we have mutated the CXXX box to obtain selectively farnesylated CNP1 or geranyl- geranylated CNP1 and found that these two modified forms of CNP1 behave identically in all of the assays performed. Isoprenylation is essential but not sufficient for the binding of in vitro synthesized CNP1 to purified myelin, because a control nonmyelin protein is isoprenylated, yet unable to bind to myelin. In our assay, membrane-bound CNP1 partitions quantitatively into the nonionic detergent-insoluble phase of myelin, suggesting that CNP1 binds to cytoskeletal elements within myelin. However, isoprenylated CNP1 fails to bind to the cytoskeletal matrix isolated from myelin by detergent treatment, implying that both detergent-soluble and insoluble myelin components are involved in the binding of CNP1. A model for the interactions between CNP1 and myelin is presented, consistent with models proposed for other isoprenylated proteins.
利用体外合成的多肽和纯化的髓磷脂，对2',3'-环核苷酸3'-磷酸二酯酶同工型1（CNP1）与髓磷脂的结合及其与髓鞘细胞骨架成分的关联进行了表征。我们之前已经表明，CNP1羧基末端CXXX框中存在的半胱氨酸残基会发生异戊二烯化，并且C15法尼基和C20香叶基香叶基类异戊二烯都可以作为修饰的底物。在这里，我们对CXXX框进行了突变，以获得选择性法尼基化的CNP1或香叶基香叶基化的CNP1，发现这两种修饰形式的CNP1在所有进行的试验中表现相同。异戊二烯化对于体外合成的CNP1与纯化的髓磷脂的结合至关重要，但并不充分，因为一种对照非髓磷脂蛋白发生了异戊二烯化，但无法与髓磷脂结合。在我们的试验中，膜结合的CNP1定量地分配到髓磷脂的非离子去污剂不溶相中，这表明CNP1与髓磷脂内的细胞骨架成分结合。然而，异戊二烯化的CNP1无法与通过去污剂处理从髓磷脂中分离出的细胞骨架基质结合，这意味着去污剂可溶和不可溶的髓磷脂成分都参与了CNP1的结合。本文提出了一个CNP1与髓磷脂之间相互作用的模型，该模型与针对其他异戊二烯化蛋白提出的模型一致。
