Piva Maria B R, Suarez Eloah R, Melo Carina M, Cavalheiro Renan P, Nader Helena B, Pinhal Maria A S
Department of Biochemistry, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil.
Department of Biochemistry, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil Department of Biochemistry, Faculdade de Medicina do ABC, Santo André 09060-650, Brazil.
Glycobiology. 2015 Sep;25(9):976-83. doi: 10.1093/glycob/cwv035. Epub 2015 Jun 1.
Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.
糖胺聚糖（GAG）在组织和细胞中普遍存在。在真核细胞中，硫酸乙酰肝素（HS）最初由一种称为乙酰肝素酶-1（HPSE）的内切β-葡萄糖醛酸酶降解。HPSE作用产生的HS寡糖增强了信号分子的活性，激活炎症反应、肿瘤转移和血管生成。本研究的目的是了解硫酸化GAG是否能调节HPSE，因为调节HPSE的机制尚未完全明确。用4-甲基伞形酮（4-MU）和氯酸钠处理CHO-K1细胞，分别促进GAG合成的完全抑制和减少硫酸化模式。在[（35）S]-硫酸盐标记后，测定野生型CHO-K1细胞和木糖基转移酶缺陷的CHO-745细胞的GAG谱。通过实时定量聚合酶链反应测定HPSE表达。用4-MU完全消除CHO-K1细胞中的GAG可抑制HPSE表达，而缺乏硫酸化则无影响。有趣的是，在这些实验中，4-MU对CHO-745细胞没有影响。此外，与主要在细胞外基质中呈现HPSE的CHO-K1野生型细胞相比，在细胞质中发现的CHO-745突变细胞中观察到了不同的酶定位。鉴于我们的结果，我们可以得出结论，GAG是HPSE表达和定位的重要调节因子。因此，GAG谱可能会影响由HPSE调节介导的细胞行为。
