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High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status

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Abstract

Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac2-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V max of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.

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Abbreviations

ABTS:

2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid

AcCoA:

acetyl-coenzyme A

ALL:

acute lymphoblastic leukaemia

AML:

acute myelogenous leukaemia

BM:

bone marrow

BSA:

bovine serum albumin

BSM:

bovine submandibular gland mucin

CML:

chronic myeloid leukaemia

CHAPS:

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

CMP-Neu5Ac:

cytidinmonophosphate N-acetylneuraminic acid

CoA:

coenzyme A

cyt-μ:

cytoplasmic μ

Cy5:

cyanin 5

de-O-AcBSM:

de-O-acetylated BSM

DAB:

3,3′-diamino benzidine

DMB:

1,2-diamino-4,5-methylene-dioxybenzene

ELISA:

enzyme linked immunosorbent assay

FACS:

fluorescence-activated cell sorter

FCS:

fetal calf serum

FITC:

fluorescein isothiocyanate

GD3:

ganglioside GD3

HPLC:

high-performance liquid chromatography

HRP:

horse radish peroxidase

IgG:

immunoglobulin G

Mab:

monoclonal antibodies

GalNAc:

N-acetylgalactosamine

Neu5,7Ac2 :

N-acetyl-7-O-acetylneuraminic acid

Neu5,8Ac2 :

N-acetyl-8-O-acetylneuraminic acid

Neu5,9Ac2 :

N-acetyl-9-O-acetylneuraminic acid

Neu5,9Ac2-GPs:

O-acetylated sialoglycoproteins

PCA:

perchloric acid

PBMC:

peripheral blood mononuclear cells

PE:

phycoerythrin

Sia:

sialic acids

SOAT:

sialate-O-acetyltransferase

SD:

standard deviation

SIg:

surface membrane Ig

TdT:

terminal deoxynucleotidyl transferase

TLC:

thin-layer chromatography

TBS:

Tris-buffered saline

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Acknowledgement

Mr. Chandan Mandal and Ms. Suchandra Chowdhury are recipients of Senior Research Fellowships of the University Grant Commission, Government of India. This work received financial support from the Department of Science and Technology, Department of Biotechnology, Indian Council of Medical Research, Council of Scientific and Industrial Research, New Delhi, India. Our sincere thanks go to Prof. Bruno Venerando (Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy) for providing the ALLPO cell line and to Dr. Bernhard Kniep (Institute of Immunology, Technical University of Dresden, Germany) for ganglioside standards. We are thankful to Prof. Tamás Laskay (Institute for Medical Microbiology and Hygiene, University of Lübeck, Germany) for providing laboratory facility to culture the T-ALL cell line. Mr. Arup Sarkar, Dr Anil.K. Chava and Mr. Asish Mallick are also acknowledged for their help. The Sialic Acids Society, Kiel, Germany, also supported this work.

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Authors and Affiliations

  1. Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, 700 032, India

    Chandan Mandal, Suchandra Chowdhury & Chitra Mandal

  2. Haematology, Kothari Medical Center, 8/3, Alipore Road, Kolkata, 700027, India

    Sarmila Chandra

  3. Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700032, India

    Chhabinath Mandal

  4. Biochemisches Institut, Christian-Albrechts Universität, Olshausenstr. 40, 24098, Kiel, Germany

    G. Vinayaga Srinivasan & Roland Schauer

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  1. Chandan Mandal
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  2. G. Vinayaga Srinivasan
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  3. Suchandra Chowdhury
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  7. Chitra Mandal
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Correspondence to Chitra Mandal.

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Mandal, C., Srinivasan, G.V., Chowdhury, S. et al. High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status. Glycoconj J 26, 57–73 (2009). https://doi.org/10.1007/s10719-008-9163-3

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