Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Abstract

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-α2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac₂-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac₂-GPs_ALL and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac₂-GPs_ALL were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac₂-GPs_ALL was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac₂-GP_ALL antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac₂-GPs_ALL as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac₂-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac₂-GPs_ALL as an alternate tool for detection of anti-Neu5,9Ac₂-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.