The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifs rationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases.

中文翻译：

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人类 TMTC1、TMTC2、TMTC3 和 TMTC4（来自 GT-C/PMT 家族的新 O-甘露糖基转移酶）中的保守序列基序被合理化为配体结合位点

人类蛋白质 TMTC1、TMTC2、TMTC3 和 TMTC4 已被实验证明是新的 O-甘露糖基化途径的组成部分。人们怀疑它们自身的甘露糖基转移酶活性，但它们的实际酶潜力尚未得到证实。到目前为止，TMTC 的序列分析已受到其膜嵌入 N 端区域内的进化序列差异、蛋白质数据库中的序列不准确以及难以解释已知同源蛋白质（主要是糖转移酶和一些具有已知的 3D 结构）。TMTC 中的进化保守分子功能只有在其膜嵌入 N 端区域内的保守膜拓扑结构下才可能实现，从而导致同源长间歇环放置在同一膜侧。使用这个标准，我们证明所有 TMTC 都有 11 个跨膜区域。与 Pfam 模型 DUF1736 同源的序列片段实际上只是 TM7 和 TM8 之间的一个环，位于 ER 腔中，包含一个小的疏水性但不包含膜嵌入的螺旋。TMTC 的膜嵌入 N 端区域不仅与 GT-C 糖转移酶亚组具有共同的折叠和 3D 结构相似性。在经过酶学和结构充分研究的 GT-C 和 TMTC 序列中，对催化、二价金属离子的结合和脂质连接糖部分的磷酸基团的结合至关重要的残基的保守性表明 TMTC 实际上是糖转移酶。我们提出了所有四种 TMTC（源自其最接近的已知同源物 5ezm/5f15）的可信 3D 结构模型，并发现观察到的保守序列基序被合理化为金属离子和多甘基-磷酸-甘露糖部分的结合位点。根据仔细的序列分析和结构建模的结果，我们可以结论性地说，TMTC 是属于 GT-C/PMT 超家族的具有酶活性的糖转移酶。DUF1736 片段是 TM7 和 TM8 之间的环，对于催化和脂质连接的糖部分结合至关重要。结合现有的间接实验数据，我们得出结论，TMTC 不仅是高等真核生物内质网中 O-甘露糖基化途径的一部分，而且实际上它们是所寻找的甘露糖基转移酶。