Mengeling B J, Manzella S M, Baenziger J U
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):502-6. doi: 10.1073/pnas.92.2.502.
The glycoprotein hormone N-acetylgalactosaminyltransferase is responsible for synthesis of Asn-linked oligosaccharides terminating with GalNAc-4-SO4 on lutropin, thyrotropin, and the uncombined glycoprotein hormone alpha subunit. We previously established that a recognition determinant for the N-acetylgalactosaminyltransferase is contained within a 22-amino acid glycopeptide fragment of the alpha subunit. We proposed that the tripeptide Pro-Leu-Arg is an essential element of the recognition determinant. Using site-directed mutagenesis we have examined the role of individual amino acids in recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase. Within the sequence Pro40-Leu41-Arg42-Ser43-Lys44-Lys45, Lys44, and Lys45, as well as Arg42 of the tripeptide, are essential for recognition. Substitution of the Leu41 with other amino acids can either increase or decrease the rate of GalNAc transfer over an 8-fold range, suggesting that the middle amino acid of the tripeptide plays a modulatory role in recognition. The critical Leu41-Arg42 and Lys44-Lys45 residues are present on the same surface of an alpha-helix, which projects from the surface of the alpha subunit. Our results indicate that an essential element of the recognition determinant consists of a cluster of basic residues and that neutral but not negatively charged residues are tolerated within this cluster.
糖蛋白激素N-乙酰半乳糖胺基转移酶负责合成在促黄体激素、促甲状腺激素及未结合的糖蛋白激素α亚基上以GalNAc-4-SO4结尾的N-连接寡糖。我们先前已确定，N-乙酰半乳糖胺基转移酶的识别决定簇包含在α亚基的一个22个氨基酸的糖肽片段中。我们提出三肽Pro-Leu-Arg是识别决定簇的一个关键元件。利用定点诱变，我们研究了单个氨基酸在糖蛋白激素N-乙酰半乳糖胺基转移酶识别过程中的作用。在Pro40-Leu41-Arg42-Ser43-Lys44-Lys45序列中，三肽的Lys44、Lys45以及Arg42对于识别至关重要。将Leu41替换为其他氨基酸可使GalNAc转移速率在8倍范围内增加或降低，这表明三肽中间的氨基酸在识别中起调节作用。关键的Leu41-Arg42和Lys44-Lys45残基位于从α亚基表面伸出的α螺旋的同一表面上。我们的结果表明，识别决定簇的一个关键元件由一簇碱性残基组成，并且该簇内可容忍中性而非带负电荷的残基。
