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

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-Oacetylneuraminic acid-a2-6-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-GPsALL and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac2- GPsALL were affinity-purified, and three distinct leukemiaspecific molecular determinants (135, 120, and 90 kDa) were demonstrated bySDS–PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac2-GPALL antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac2-GPsALL 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 (n¼50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma as well as normal healthy individuals (n¼21). This study demonstrated the potential of purified Neu5,9Ac2-GPsALL as an alternate tool for detection of anti-Neu5,9Ac2-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmani- asis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid- binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both a2,3- and a2,6-linked sialic acids. No endo- genous biosynthetic machinery for Neu5Ac could be demon- strated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demon- strated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite mem- brane and culture medium showed three analogous sialogly- cans corresponding to 130, 117, and 70 kDa, indicating that a2,3- and a2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in a2!6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytome- try, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialo- glycans. Our results indicate that sialic acids (a2!6 and a2!3 linked) and 9-O-acetyl derivatives constitute compo- nents of the parasite cell surface

The Glycome of Normal and Malignant Plasma Cells

<div><p>The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10) and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i) malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii) be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii) Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14), t(4;14), hyperdiploidy, 1q21-gain and deletion of 13q14. iv) A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v) As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.</p></div

Upregulated genes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways.

<p>Using the Goeman's global test method the degree of distinction between myeloma cell samples (MM) and normal bone marrow plasma cell (BMPC) samples was investigated. The significance of p-level indicates the degree of difference between then BMPC and the MM samples. Glycan pathways were ordered according to the general families of macromolecular glycan structures, i.e. GPI-anchored glycoproteins, N- and O-glycosylated glycoproteins, glycosphingolipids (GSL) and proteoglycans (heparan sulfate (GAG-HS) glycosaminoglycans (GAG); chondroitin sulfate (GAG-CS) and keratin sulfate (GAG-KS). Most significant changes in the MM glycome were found in the GPI- and in the HS synthesis and were highlighted.</p

Steps of heparan sulfate and GPI anchor biosynthesis.

<p><b>A</b>: Biosynthetical steps of the GAG-HS pathway and the functional role of the enzymes are shown. Green arrows indicate upregulated gene expression and red arrows downregulated gene expression. <b>B</b>: The steps of the GPI anchor biosynthesis are shown and the function of the proteins corresponding to the overexpressed <i>PIG</i>-genes M, C, B are shown.</p

Gene expression of significantly overexpressed genes related to the glycosylphosphatidylinositol (GPI) anchor pathway.

<p><b>A–C:</b> Median gene expression of phosphatidylinositol glycan anchor biosynthesis, class (<i>PIG</i>) -M, - C, B is significantly higher in malignant vs. normal bone plasma cells. Boxplots display the median and range for these genes for myeloma patients categorized into Durie-Salmon stage I-III, myeloma is compared to results in normal bone marrow plasma cells (ND), and human myeloma cell lines (HMCL). <b>D/E:</b> Validation of <i>PIG-M</i> gene expression using RT-PCR and Western Blotting using the HMCL OPM-2 with a high PIG-M expression and the HMCL KMS-12-BM with a low PIG-M expression as per gene expression profiling. <b>D:</b> mRNA expression of <i>PIG-M</i> gene was investigated using RT-PCR. <b>E:</b> Western Blotting was performed using standard markers tubulin and PDI, a marker for endoplasmatic reticulum, to confirm equal loading of gel and investigate the PIG-M protein of OPM-2/PIG-M expression high (left lane) and the KMS-12-BM/PIG-M expression low (right lane). <b>F:</b> Flow cytometric analysis of CD55 and CD56 on KMS-12-BM and OPM-2 myeloma cell lines with and without prior PLC treatment. Red lines: CD55/CD59 expression without PLC treatment, blue lines: CD55/CD59 expression after PLC-treatment, green lines: background staining solely with secondary antibody coupled to FITC.</p

<i>PIGM</i> expression correlated to 1q21 copy number.

<p><i>PIGM</i> gene expression was plotted as log 10 and correlated with cytogenetically determined 1q21 gene copy number.</p

Gene expression of significantly regulated genes of the heparan sulfate pathway.

<p><b>A–D</b>: Four genes of the glycosaminglycan-heparan sulfate (GAG-HS) biosynthetic pathway (<i>EXT2/EXTL2/B3GAT1/HS2ST1</i>) are significantly upregulated in myeloma compared to normal bone marrow plasma cells. Boxplots display the median and range for these genes for myeloma patients categorized into Durie-Salmon (DS) stage I-III (MM1-3), bone marrow plasma cells from normal donors (ND), and human myeloma cell lines (HMCL). <b>E,F</b>: Two heparan sulfotransferases (<i>HS6ST1</i> and <i>HS3ST1</i>) of the GAG-HS biosynthetic pathway are significantly downregulated in myeloma compared to normal plasma cells. Boxplots display the median and range for these genes for myeloma patients categorized into Durie-Salmon stage I-III (MM1-3), normal bone marrow plasma cells (ND), and human myeloma cell lines (HMCL).</p