Blanz Judith, Zunke Friederike, Markmann Sandra, Damme Markus, Braulke Thomas, Saftig Paul, Schwake Michael
Biochemisches Institut der Christian Albrechts Universität zu Kiel, Otto-Hahn-Platz 9, 24118, Kiel, Germany.
Arbeitsbereich Molekularbiologie, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
Traffic. 2015 Oct;16(10):1127-36. doi: 10.1111/tra.12313. Epub 2015 Sep 1.
The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for β-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo.
溶酶体整合膜蛋白2（LIMP-2/SCARB2）已被描述为β-葡萄糖脑苷脂酶（GC）的一种不依赖甘露糖6-磷酸（M6P）的转运受体。最近，在重组表达的LIMP-2胞外域的晶体结构中报道了一个假定的M6P残基。基于表面等离子体共振和荧光寿命成像分析，有人提出可溶性LIMP-2与不依赖阳离子的M6P受体（MPR）的相互作用导致LIMP-2以M6P依赖的方式靶向溶酶体。由于未探讨这一观察结果的生理相关性，我们研究了LIMP-2在小鼠肝脏和小鼠胚胎成纤维细胞中以M6P依赖的方式向溶酶体的递送情况。我们证明LIMP-2和GC到达溶酶体不依赖于M6P途径。在缺乏MPR或形成M6P的N-乙酰葡糖胺（GlcNAc）-1-磷酸转移酶的成纤维细胞中，LIMP-2仍定位于溶酶体。免疫印迹分析还显示，从野生型（wt）和GlcNAc-1-磷酸转移酶缺陷型小鼠肝脏中纯化的溶酶体内LIMP-2水平相当。LIMP-2腔结构域在野生型、LIMP-2缺陷型和GlcNAc-1-磷酸转移酶缺陷型细胞中的异源表达进一步证实，M6P修饰对于LIMP-2的溶酶体分选是可有可无的。最后，组织蛋白酶Z是一种已知的GlcNAc-1-磷酸转移酶底物，但不是LIMP-2，可被M6P特异性抗体沉淀。这些数据证明了LIMP-2以及随后的GC在体内以不依赖M6P的方式进行溶酶体分选。
