Hieronimus Bettina, Pfohl Julian, Busch Christian, Graeve Lutz
Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.
Section of Dermatolo-Oncology, Department of Dermatology and Allergology, University of Tuebingen, Tuebingen, Germany.
Cell Physiol Biochem. 2017;42(1):198-210. doi: 10.1159/000477311. Epub 2017 May 25.
BACKGROUND/AIMS: Membrane-type matrix metalloproteinases (MT-MMPs) are expressed on the cell surface and hydrolyze extracellular matrix components and signaling molecules by which they influence cancer cell migration and metastasis. Two of the six known MT-MMPs are anchored to the plasma membrane via a GPI anchor, one of which is MT4-MMP. Only little is known about MT4-MMP expression, synthesis, regulation and degradation.
We analyzed several human cancer cell lines as well as tissue homogenates using Western blotting and quantitative PCR for the expression of MT4-MMP. Organelles of SK-Mel-28 cells were separated using continuous Iodixanol gradients. Glycosylation of the SK-Mel-28 protein was studied via glucosidases and site directed mutagenesis of the MT4-MMP cDNA prior to transfection.
We found the MT4-MMP highly expressed in human melanoma cell lines as well as skin and melanoma tissue samples. Three forms of MT4-MMP with molecular masses of 45 kDa, 58 kDa and 69 kDa were detected. Further, we demonstrate that the 58 kDa form is the mature protein in the cell membrane, while the 69 kDa form is its precursor found in intracellular compartments. The 69 kDa forms are processed by furin cleavage in the Golgi apparatus. Moreover, we identified Asn318 as the single N-glycosylation site of MT4-MMP.
We demonstrate the novel expression of MT4-MMP in melanocytic tissues and propose a precursor/product-relationship of the different forms of MT4-MMP in melanoma cells.
背景/目的：膜型基质金属蛋白酶（MT-MMPs）在细胞表面表达，可水解细胞外基质成分和信号分子，进而影响癌细胞的迁移和转移。已知的六种MT-MMPs中有两种通过糖基磷脂酰肌醇（GPI）锚定在质膜上，其中之一是MT4-MMP。目前对MT4-MMP的表达、合成、调控及降解了解甚少。
我们使用蛋白质免疫印迹法和定量PCR分析了几种人类癌细胞系以及组织匀浆中MT4-MMP的表达。通过连续的碘克沙醇梯度分离SK-Mel-28细胞的细胞器。在转染前，通过葡萄糖苷酶和MT4-MMP cDNA的定点诱变研究SK-Mel-28蛋白的糖基化。
我们发现MT4-MMP在人类黑色素瘤细胞系以及皮肤和黑色素瘤组织样本中高表达。检测到三种分子量分别为45 kDa、58 kDa和69 kDa的MT4-MMP形式。此外，我们证明58 kDa形式是细胞膜中的成熟蛋白，而69 kDa形式是在细胞内区室中发现的其前体。69 kDa形式在高尔基体中通过弗林蛋白酶切割进行加工。此外，我们确定Asn318是MT4-MMP的单一N-糖基化位点。
我们证明了MT4-MMP在黑素细胞组织中的新表达，并提出了黑色素瘤细胞中不同形式的MT4-MMP的前体/产物关系。
