Purpose: To study the structural alterations of asparagine-linked sugar chains (N-glycans) on urine fibronectin (Fn) from bladder cancer (BCa) patients and its enzymatic mechanism.
Methods: Eight pairs of urine samples from eight BCa patients pre-operation and 3 months post-operation (which proved to be normal) were collected, and the Fn in the urine samples was purified with an anti-Fn antibody affinity column. Different lectins labeled with horseradish peroxidase (HRP) were used as probes to bind the glycans of purified Fn immobilized on membrane. Enhanced chemiluminescence (ECL) reagent was adopted to estimate the activity of the bound HRP as a measure of the binding affinity of the Fn glycans to lectins, and expressed as luminescent light units (LLU). The enzymatic mechanism of the structural alteration of N-glycans in BCa Fn was studied by determination of GnT activities using the HPLC method and fluorescent-labeled substrate.
Results: The mean LLU of BCa Fn was only 18.1% of the normal samples when Con A-HRP were used as probes, while the mean LLU of the BCa group was 3.34 times and 3.26 times higher than normal for the DSA-HRP and WGA-HRP probes, respectively. The individual data of the patients did not overlap between the BCa sample and normal counterparts, indicating that the positive rates were 100%, regardless of which lectin-HRP was used. These results reveal that the antennary number and bisecting GlcNAc structure are increased in the N-glycans of urine Fn from BCa assessed according to the binding specificity of ConA, DSA, and WGA. In addition, the binding affinities of urine Fn with DSA and WGA were correlated to pathological stage, and the affinity of Fn with WGA was also correlated with pathological grade. The results of GnT determination showed that GnT-III, IV, and V in BCa tissues increased by 34.0, 18.1, and 1.6 times, respectively, in normal bladder tissues which were at least 5 cm away from the BCa of the same bladder. These findings were compatible with the structural changes of N-glycans in BCa Fn, since GnT-III and GnT-IV/V are responsible for the synthesis of bisecting GlcNAc and the increase of antennary number in N-glycans, respectively.
Conclusions: (1) The highest elevation of GnT-III and the close relationship between the WGA binding of BCa Fn with the pathological stage and grade of BCa indicate that the increase of bisecting GlcNAc in N-linked glycans contributes more to the malignant behavior of BCa than the increase of GnT-IV, GnT-V, and the antennary number. (2) The correlation of altered activities of bladder GnTs with the abnormal structures of urine Fn in BCa patients indicates that the urine Fn is synthesized in the bladder. (3) The lectin-HRP assay for analyzing the structure of N-glycans in urine Fn may be used as a simple and accurate diagnosis method for BCa in the future.