Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum

Abstract

Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.

Keywords: Cancer serum; diagnostic marker; ganglioside; prostate cancer; sialidase.

Fig 1

Determination of ganglioside sialidase activity in human serum and increase in the serum of prostate cancer patients. (a) Note significantly higher levels of sialidase activity toward the ganglioside GM3 substrate in serum of prostate cancer patients than in normal subjects. (b) Statistical significance of increased sialidase activity in the serum of prostate cancer patients compared with normal subjects.

Fig 2

Characterization of a ganglioside sialidase activity in human serum. (a) Sialidase activity toward GM3 gangliosides with various serum amounts. Note that with 5 μL of serum decreased sialidase activity was observed. (b) Effect of pH on the sialidase activity. Sialidase activitity was assayed using GM3 substrate in sodium acetate buffer at pH 3.5–5.5 and sodium phosphate buffer at pH 5.5–7.0. (c) Substrate specificity of the serum sialidase. Various natural substrates and a synthetic substrate, 4MU-NeuAc, were examined. (d) Immunoprecipitation studies of the serum sialidase using anti-NEU3 and NEU4 antibodies. Anti-NEU3 and anti-NEU4 antibodies precipitated recombinant NEU3 (left panel) and NEU4 (middle), respectively. The serum sialidase was precipitated only with anti-NEU3 antibody (right panel).

Fig 3

Characterization of the inhibitor activity in human serum. (a) Inhibitory effects of human serum on ganglioside sialidase activity. The inhibitory activity was defined as the percentage of residual sialidase activity relative to the controls without inhibitor. Ammonium sulfate fractions of whole serum were examined for inhibitory activity against recombinant NEU3 or NEU4 sialidase activity (0.8–1.2 units of each) with the GM3 substrate. The results here were obtained only with NEU3 but not with NEU4. (b) The 50–80% ammonium sulfate fractions were tested for inhibitory activity against recombinant sialidase (0.9–1.3 units of each) NEU3 or NEU4 with ganglioside GM3 (left panel), and NEU1, NEU2 and NEU4 with 4MU-NeuAc as the substrate (right panel). (c, d) Immunoprecipitation studies of the serum inhibitory activity with anti-human albumin goat antibody (Bethyl Laboratories, Montgomery) (c) or protein L-Sepharose (1.5 × 3 cm, BioVision Inco.) (d). As controls, goat IgG and Sepharose beads were used instead of anti-albumin antibodies and protein L-Sepharose, respectively. Recombinant NEU3 (1.5 units) was preincubated with human serum (5 μL) or serum treated with anti-albumin antibodies (c) or protein L-Sepharose (d), and each supernatant after immunoprecipitation was assayed for sialidase activity with GM3 as the substrate.

Fig 4

Separation of the sialidase from the inhibitory activities. (a) Separation of the two activities by exosome isolation kit (ExoQuick, System Biociences). Resulting supernatants after exosome isolation retained most of the sialidase activity, whereas the exosome fraction had enriched inhibitory activity. The western blot with anti-HSP70 antibody is shown for each exosome fraction to verify the fractionation. (b) Separation of the two activities on Octyl-Sepharose chromatography. Ammonium sulfate fractions (40–80%, containing sialidase and inhibitory activities) of cancer patient serum (3 mL) were dissolved in 0.1 M phosphate buffer (pH 6.8) containing 0.5 M NaCl and 0.2 mM PMSF, and loaded on a Octyl-Sepharose column (1.5 × 3 cm, GE health care) equilibrated with the same buffer. After washing the column with the same buffer, the sialidase activity was eluted with 10 mM phosphate buffer containing 0.2 mM PMSF, and then the inhibitory activity followed with 0.2% Triton X-100 in the buffer. (c,d) The each activity fractions from the Octyl-Sepharose column were concentrated and separately applied to the Toyopearl HW-55F column (1.5 × 45 cm, Tosoh Bioscience) to estimate the apparent molecular size. BSA, bovine serum albumin.

Fig 5

Sialidase NEU3 assay by ELISA. (a) Standard curve of sialidase NEU3 concentrations using recombinant NEU3 protein in the sandwich ELISA. Homogenates of vector-transfected and NEU3-transfected 293T cells were used as the control and NEU3 standard, respectively. (b) The results of increased sialidase NEU3 in the serum of prostate cancer patients (n = 24) compared with normal sera (n = 9) as assessed by the ELISA method.