Extra-Lysosomal Localization of Arylsulfatase B: Implications for Epithelial Pathobiology
SV Prabhu, G Guzman, S Bhattacharyya, AK Balla, JK Tobacman. University of Illinois at Chicago, Chicago, IL; Jesse Brown VA Medical Center, Chicago, IL
Background: Arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase) is deficient in MPS VI, the lysosomal storage disease characterized by accumulation of chondroitin-4-sulfate and dermatan sulfate. Recent work demonstrated reduction of ASB activity in patients with cystic fibrosis and in malignant mammary epithelial cell lines. In colonic epithelial cell lines, reduced ASB activity was associated with increases in cell migration, RhoA activation and MMP9 expression. Surprisingly, immunohistochemistry (IHC) and activity assays demonstrated ASB in cell membranes of cultured human bronchial and colonic epithelial cells, as well in the cytoplasm and nuclei.
Design: To further assess localization of ASB in normal and malignant colonic epithelial cells, we measured ASB activity using the substrate 4-MUS in a quantitative fluorometric assay. IHC of ASB was assessed in 75 cores in tissue microarrays from the National Disease Research Interchange (Philadelphia, PA). A rabbit polyclonal antibody (Open Biosystems, Huntsville, AL) directed at a unique C-terminal epitope and shown to be specific for ASB was used for IHC. Three observers independentlly reviewed each of the sections, assessing % positivity and intensity of cytoplasmic, nuclear, and luminal membrane staining.
Results: In normal colonic tissue, IHC demonstrated marked prominence of ASB in the luminal membrane. In malignant tissue, luminal membrane staining was reduced or absent. Increased cytoplasmic intensity was apparent near the luminal surface in the normal tissue, in contrast to deeper in the crypts. This variation was absent in the malignant cells. In the normal tissue, rare, isolated cells, with intense, uniform cytoplasmic and nuclear staining, were dispersed in the crypts. Nuclear staining was generally absent in the malignant epithelial cells; when present, staining was punctate. Activity assay demonstrated that ASB activity was significantly less in malignant colonic tissue than in normal tissue (56.3 ± 4.0 vs. 108.1 ± 7.8 nmol/mg protein/hr; p<0.0001, unpaired t-test, two-tailed, n=6).
Conclusions: The enzyme ASB is not confined to lysosomes in colonic epithelial cells, but also has luminal membrane localization. The distinct cellular pattern of ASB staining in the normal colonic epithelial cells is lost in the malignant tissue, and ASB activity is significantly reduced. ASB may be a useful marker of colonic pathology.
Wednesday, March 24, 2010 9:30 AM
Poster Session V # 216, Wednesday Morning