GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.
GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.
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DOI：
10.7554/elife.08916
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发表时间：
2015-09-15
期刊：
影响因子：
7.7
通讯作者：
Stanley P
中科院分区：
文献类型：
--
作者：
Huang HH;Hassinen A;Sundaram S;Spiess AN;Kellokumpu S;Stanley P
Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis. DOI: http://dx.doi.org/10.7554/eLife.08916.001 Proteins are made up of chains of amino acids that fold into three-dimensional shapes and many are assembled in a cell compartment known as the endoplasmic reticulum. From here, these new proteins move to another compartment called the Golgi, where they may be further modified before they are transported to their final destination in the cell. One way that proteins may be modified is known as glycosylation, in which sugar molecules are attached to specific amino acids. Some sugar molecules can act as labels that ensure the new proteins are transported to the correct destination in the cell. For proteins that are delivered to the surface of the cell, the sugar molecules can also play important roles in communication with other cells. A simple sugar molecule, or a complex arrangement of many sugar molecules, may be attached to an amino acid by glycosylation. An enzyme called MGAT1 controls the synthesis of sugars called complex N-glycans in the Golgi. In 2010, researchers reported that a glycoprotein called GnT1IP-L binds to MGAT1 and inhibits its activity, thereby blocking the production of complex N-glycans. GnT1IP-L was found in the endoplasmic reticulum and Golgi, but it was not clear how it inhibits MGAT1. Huang et al.—including some of the researchers from the 2010 study—have now investigated the activity of GnT1IP-L in cells grown in the laboratory using several biochemical techniques. The experiments show that GnT1IP-L only binds to MGAT1 when both proteins are in the Golgi. There are three sections (or ‘domains’) in GnT1IP-L, but Huang et al. found that only the domain that is on the inside of the Golgi is involved in this interaction. Previous work indicated that GnT1IP-L may be involved in the formation of sperm in mice. Huang et al. have now analyzed previously published data on samples of testis tissue from human patients and found that the gene that encodes GnT1IP-L is present in very low amounts in patients whose sperm do not develop properly. Huang et al.'s findings suggest that GnT1IP-L may inhibit MGAT1 to control the glycosylation of proteins in the Golgi of developing sperm. The next step is to test this hypothesis by generating mutant mice that lack GnT1IP-L, or to make GnT1P-L in other cells in which it is not normally made, to find out if this affects the production of sperm. DOI: http://dx.doi.org/10.7554/eLife.08916.002
