α-N-acetylgalactosaminidase: a potential cellular immunodeficiency factor in cancer patients

Objectives: The present study aimed to determine if human salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase (α-NaGalase) affects the bioactivity of an O-linked macrophage-activating factor (GcMAF) that is important for immunity.

Materials and Methods: The Exo-α-NaGalase activity in the human salivary gland adenocarcinoma cell line (HSG), squamous cell carcinoma cell line (SCCTM), normal keratinocytes and fibroblasts was determined spectrophotometrically. The HSG-α-NaGalase was purified by ion exchange and gel filtration chromatography using an exo-α-NaGalase activity assay as an indicator of fractionation. HSG-α-NaGalase enzyme activity was characterized in an in vitro deglycosylation assay using peanut agglutinin lectin. The activity of GcMAF was assayed in a superoxide generation assay and by determination of its effect on monocyte / macrophage phagocytic activity.

Results: Higher levels of exo-α-NaGalase activity were detected in cell extracts and the conditioned medium of the HSG adenocarcinoma cell line compared to the SCCTM squamous cell carcinoma cell line. Normal keratinocytes and fibroblasts exhibited low levels of exo-α-NaGalase activity. Characterization of purified HSG-α-NaGalase indicated that the enzyme hydrolyzes synthetic substrates and displays low intrinsic endo-α-NaGalase and α-galactosidase activities in addition to its exo-α-NaGalase activity. Pre-treatment of GcMAF with HSG-α-NaGalase decreased its ability to activate monocyte / macrophage superoxide-generation compared to non-treated GcMAF. Furthermore, unlike non-treated GcMAF, the pre-treated GcMAF was unable to enhance the phagocytic activity of monocytes / macrophages. An in vitro deglycosylation assay of GcMAF, using peanut agglutinin lectin which recognizes galactose / N-acetylgalactosamine residues, demonstrated that HSG-derived α-NaGalase displays both exo- and endo-enzyme activities and facilitates sialidase activity.

Conclusion: These results demonstrate that HSG-derived α-NaGalase possesses unique enzymatic properties when compared to the enzyme in normal cells, and is a candidate factor for contributing to cellular immunodeficiency in the GcMAF-related immune cascade for host defense in cancer patients with salivary gland adenocarcinomas.