Investigation of 9‐<i>o</i>‐acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (2000)

Modern treatment protocols of childhood acute lymphoblastic leukaemia (ALL) utilize the biological and clinical features of the disease to tailor the intensity of therapy to the risk of relapse by evaluation of minimal residual disease (MRD) using several molecular techniques. To minimize the likelihood of false negative results, tandem application of at least two methods for detection of MRD is currently recommended. To address this question, the present review deals with the recently evolved information reported by our group regarding the presence of O-acetylated sialoglycoconjugates (O-AcSGs) in childhood ALL and their potential as novel biomarkers for monitoring the disease. Sialic acids are a family of N- and O-substituted derivatives of neuraminic acid, commonly referred to as N-acetyl neuraminic acid and abbreviated as Neu5Ac ( Schauer, 1982). It is the most abundantly present monosaccharide as the terminal residue of vertebrate cell-surface sugar chains. The accessibility provided by its strategic terminal position is reflected in the regulation of a multitude of cellular and molecular interactions ( Varki, 1992; Kelm & Schauer, 1997; Schauer et al, 1997 ) . In recent years, steady refinement of analytical techniques has enabled glycobiologists to undertake detailed mapping of this structural diversity ( Schauer et al, 1995 ). Among the diverse multitude of variations of the parent sugar Neu5Ac, the most frequently occurring modification is O-acetylation at position C-7,8,9 to form N-acetyl-7,8,9-O-acetyl neuraminic acid, respectively, leading to a family of O-acetylated sialoglycoconjugates or O-AcSGs ( Shi et al, 1996a; Klein & Roussel, 1998). However, as O-acetyl esters from C-7 and C-8 positions can, even under physiological conditions, spontaneously migrate to the C-9 position, O-acetylation at the C-9 position is the most common biologically occurring modification ( Varki, 1992, 1997). There is growing evidence to suggest that a number of physiological and pathological processes such as cell–cell adhesion, signalling, differentiation and metastasis may be attributed to the appearance of O-AcSGs ( Varki, 1992). Accordingly, study of their occurrence, metabolism and quantitative and qualitative analysis would provide an insight into the structure and function of these fascinating molecules. As the appearance of O-AcSGs on glycoconjugates (either glycoproteins or glycolipids) is cell-type specific and developmentally regulated, it is expected that their synthesis and turnover will be a finely tuned phenomenon ( Schauer, 1991). After the translocation of cytidine monophosphate (CMP)-sialic acid residues into the golgi apparatus or the endoplasmic reticulum, sialyltransferases present in the golgi catalyse their transfer onto an appropriate acceptor molecule. Usually, the concerned acceptor structure is an oligosaccharide chain of a nascent glycoconjugate to which sialic acid can be linked in an α2→3, α2→6, α2→8 or α2→9 linkage with the accepting subterminal sugar, which is usually galactose (Gal) or N-acetyl galactosamine (GalNAc) or less commonly N-acetyl glucosamine (GlcNAc). Subsequently, the sialoglycoconjugate generated may be further modified by O-acetyl transferases. These enzymes transfer the acetyl group from acetyl-coenzyme A (CoA) onto sialoglycoconjugates at the C-7,8,9 positions, thereby generating O-acetylated sialoglycoconjugates or O-AcSGs ( Fig 1; Varki, 1997; Traving & Schauer, 1998). The primary insertion site for the O-acetyl group may well be the C7-OH group, from where it can non-enzymatically migrate to the C-9 position, presumably via C-8, leaving the C7-OH group available for a new transfer ( Klein & Roussel, 1998). Metabolic pathways of O-acetylated sialoglycoconjugates. Interplay of the different enzymatic pathways involved in their biosynthesis, transfer, spontaneous modification and catabolism are shown. Once attached to the sialoglycoconjugates, these O-acetyl esters need to be removed at some point in the life cycle of the parent molecule ( Fig 1). The key enzymes involved are a family of sialate 9-O-acetyl esterases followed by sialidases ( Diaz et al, 1989 ; Higa et al, 1989 ). Taken together, it appears that O-acetylation is like a see-saw, the equilibrium being maintained by the activities of sialyltransferases and sialate O-acetyl transferases at one end and the O-acetyl esterases and sialidases on the other. Therefore, one could envisage that in disease conditions any modification of these regulatory enzymes would be reflected in altered expression of O-AcSGs. In fact, the enzyme uridine diphosphate-N-acetylglucosamine 2-epimerase has been shown to be an important rate-limiting enzyme in the sialic acid biosynthetic pathway and consequently a major determinant of cell-surface glycoconjugate sialylation in haematopoietic cell lines ( Keppler et al, 1999a ). It would be interesting to study whether a similar scenario exists with regard to O-AcSGs. A word of caution is that, to date, identification and characterization of sialate O-acetyl transferases remains an intractable problem. Identification of key enzymes would definitely contribute towards a detailed understanding of disease pathogenesis and progression and would provide clues for designing new drugs. Analytical methods for quantification of O-AcSGs require their previous release from glycosidic linkages by either enzymatic or chemical hydrolysis. However, the currently available methods are unsatisfactory as potential pitfalls during analysis include incomplete release of sialic acids, de-O-acetylation and spontaneous migration of O-acetyl groups. Therefore, an urgent need exists for development of tools to detect alkali-labile O-AcSGs. Probes for the detection of sialoglycoconjugates include a wide variety of sialic acid-binding lectins from natural sources ( Mandal & Mandal, 1990; Varki, 1997). In comparison, tools to identify the presence of O-acetylated sialoglycoconjugates remain limited. Possibly the commonest probe used for detecting the presence of 9-O-AcSGs irrespective of their linkage and the subterminal sugar, is the influenza C virus ( Rogers et al, 1986 ) or its recombinant soluble form with the Fc portion of human immunoglobulin (Che-FcD) ( Zimmer et al, 1992 ; Klein et al, 1994 ). In contrast to the broad binding specificity of the influenza C virus, is a lectin achatinin H, isolated from the haemolymph of the African giant snail Achatina fulica and affinity-purified using bovine submaxillary mucin ( Sen & Mandal, 1995). This lectin shows a Ca2+-dependent binding specificity for 9-O-AcSGs in an α2→6 linkage with a specific subterminal sugar, namely GalNAc of the underlying oligosaccharide chain ( Basu et al, 1986 , 1988; Mandal & Basu, 1987; Mandal et al, 1989 ; Sen & Mandal, 1995; Table I). Accordingly, its lectinogenic epitope has been defined as 9-O-AcSA α2→6 GalNAc ( Fig 2). Another lectin obtained from the Californian coastal crab Cancer antennarius binds sialic acid O-acetylated at both the C-4 and C-9 positions ( Ravindranath et al, 1985 ). The sialic acids with their 9-carbon backbone in chair conformation. R1, R2 and R3 represent different natural substitutions. When R1 at C-9 is substituted by an O-acetyl group and is linked in an α2→6 linkage with GalNAc, it represents the lectinogenic epitope of achatinin H. In the absence of commercially available O-acetylated sialic acid-binding lectins, alternative approaches use the two plant lectins Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA); these two lectins bind to sialic acid in an α2→6 ( Shibuya et al, 1987 ) and α2→3 linkage ( Wang & Cummings, 1988), respectively, to GalNAc. Therefore, any increase in the binding of these two lectins after de-O-acetylation is evidence of the presence of O-AcSGs in a defined linkage and with a known subterminal sugar. Naturally occurring antibodies against O-AcSGs have been identified in minimal amounts in normal human serum belonging to the IgG2 subtype ( Zeng et al, 1992 ) , but their biological implications, however, still remain undefined. In disease conditions, the presence of such antibodies in significantly increased amounts has been identified by our group in two clinically diverse conditions, namely acute lymphoblastic leukaemia (ALL) ( Pal et al, 2000 ) and visceral leishmaniasis ( Chatterjee et al, 1998 ). Identification of the O-acetylated form of a neuroectoderm-associated disialoganglioside (O-AcGD3) has been discovered by using a panel of monoclonal antibodies (mAbs) ( Cheresh et al, 1984; Levine et al, 1984 ). Another O-acetylated ganglioside O-AcGT3 can be recognized by mAbs 493D4 ( Zhang et al, 1997 ) and A2B5 ( Dubois et al, 1990 ). CDw60 antigens, as O-AcGD3, present on T cells have been detected by a panel of mAbs ( Kniep et al, 1992 ). It may be noted that among all these mAbs only 493D4 can detect O-AcSGs present on murine glycoproteins ( Zhang et al, 1997 ), the remaining mAbs detecting O-AcSGs on glycolipids. Accordingly, efforts should now be aimed at development of mAbs which can detect O-AcSGs present on glycoproteins. With these caveats in mind, the probes discussed above would need to be harnessed in various permutations and combinations in order to identify specifically not only the presence of O-AcSGs but also to define their different linkages to the specified subterminal sugar of the underlying sugar chain. To achieve this, a combination of Cancer antennarius lectin, influenza C virus or CHE-FcD, achatinin H, SNA and MAA may be used to distinguish between sialoglycoconjugates having terminal 4- and 9-OAcSGs in an α2→6, α2→3 and α2→8 linkage ( Varki, 1997). O-AcSGs are developmentally regulated in various systems including the central and peripheral nervous systems, the retina, the adrenal medulla and the kidney. Possibly the best characterized is the ganglioside 9-O-acetyl GD3, which is expressed on primitive neuroectodermal cells and disappears in adult life ( Levine et al, 1984 ). It reappears as oncofetal antigens in melanomas, detected by Cancer antennarius lectin ( Ravindranath et al, 1988 , 1989) and a panel of monoclonal antibodies ( Cheresh et al, 1984; Levine et al, 1984 ). Additionally, basal and suprabasal keratinocytes of patients with psoriasis express the T-cell antigen CDw60, an O-acetyl GD3 disialoganglioside ( Skov et al, 1997 ). Similarly, of O-acetyl GD3 has also been in ( et al, ). The presence of has also been identified in and used for therapy ( et al, ; et al, 1998 ). of the sialic acids present on are usually at C-9 ( & 1989) . and have identified the presence of ( et al, ) and also and ( & of and human O-AcSGs that are developmentally regulated and increase after ( et al, 1987 ). to their to O-AcSGs are to a against by chemical and ( et al, 1992 , In pathological conditions, including ( et al, ), ( et al, 1997 ) and disease ( et al, ), a of O-AcSGs has been its to the form ( et al, ; et al, 1988 ). This in O-AcSGs is with a increase in expression of the antigens, and ( et al, 1995 ), which are to be ( et al, 1997 ). the broad binding of influenza C virus to their in has been shown on both glycoproteins and on human T and ( et al, ; Zimmer et al, 1994 ). human a ganglioside GD3 antigen has been identified ( Kniep et al, 1992 ), present on and a of T ( Kniep et al, ). In the antigen as a for cells ( et al, 1997 ). of this antigen has two in human T cells to be The T cells with the are the cell that provide to the are T cells that differentiation ( & In such as and melanomas, an increased appearance of 9-O-AcSGs on human has been the sialic acid by of which is O-acetylated at C-9 ( et al, 1988; et al, ). acute lymphoblastic leukaemia (ALL) may be defined as a characterized by of from ( 1995; & 1995). the binding of a lectin, achatinin H, towards have identified the presence of O-AcSGs on of ALL patients of both T and ( Mandal et al, 1997; et al, 1999a ). These O-AcSGs are present in the peripheral cells of normal ( et al, ). a of with normal minimal with for acute leukaemia leukaemia leukaemia and similar to normal To that specifically its lectinogenic our study two cell namely a cell known to have a of 9-O-AcSGs ( Zimmer et al, 1995 ), and a cell cell lines which that the binding of achatinin to O-AcSGs. The cell-surface of 9-O-AcSGs on the of ALL patients by using achatinin H. minimal binding in normal with its binding with both and patients ( Fig using monoclonal antibodies with specificity towards and with achatinin further that of both and T-cell O-AcSGs. Taken together, our that the 9-O-AcSGs recognized by achatinin could be as potential antigens, on however, this still to be A of analysis of binding of to peripheral cells of patients with and and a normal ( et al, 1999a ). with and for represent the of and binding to negative and To provide evidence for the presence of O-AcSGs on the of ALL the of sialic acid that is O-acetylated The in ALL patients in normal Similarly, and cell lines also a of and ( et al, 1999a ). As this the of sialic acid that O-acetylated at irrespective of the linkage or the subterminal sugar, it remains an as to whether the 9-O-AcSGs detected by achatinin and the are one and the and methods for sialic acid and its derivatives include using and a binding of achatinin with O-AcSGs from the of normal and ALL patients ( et al, 1999a ). two O-AcSGs to and However, in the ALL an two O-AcSGs to and as the binding it that achatinin specifically to O-AcSGs. Accordingly, the epitope present on of ALL patients may be to be a determinant linked to GalNAc in an α2→6 It may be that and characterization of antigens, the O-AcSGs to and would for the of for chain detection of Another that may be is the development of monoclonal antibodies against these that may be for and the presence of antibodies against O-AcSGs in normal human serum has been identified ( Zeng et al, , to has been in either the presence of such an in disease conditions or in it a biological After identification of an increased expression of cell-surface 9-O-AcSGs on the of ALL present on normal it to identify whether these and could leading to increased against significantly increased against O-AcSGs of and IgG2 identified from the serum of ALL patients ( Pal et al, 2000 ). a on information that the sialic acid abundantly present in bovine submaxillary mucin a of the O-acetyl group, ( Schauer, 1982). Accordingly, using bovine submaxillary mucin as the an for of where with patients having and to the increased presence of antibodies of IgG2 subtype in the serum of and with visceral leishmaniasis ( Chatterjee et al, the may a between the two ALL and visceral but are clinically As the of ALL patients at an with the of Pal et al, 2000 ), that this as a for the of ALL patients and clinical As treatment protocols for ALL use molecular and information to tailor the intensity of therapy to the risk of have the expression of 9-O-AcSGs in patients under different of treatment to its with the disease After the of a between the presence of O-AcSGs on the of ALL patients at used the potential of the lectin achatinin to a for the clinical of ALL the involved of the with different of the lectin achatinin for The cells with and any as a of cell ( et al, ; Mandal et al, 1997 ). can be substituted with a ( Chatterjee et al, 1990 the cell is to the of by the enzymes ( et al, ). the of achatinin that as the It is from Fig that the of achatinin in both a and at only This the increased expression of 9-O-AcSGs on irrespective of their In normal a of achatinin to It may be noted that in normal not only the of achatinin significantly but also the of that in and patients ( Mandal et al, 1997 ; et al, ). A of the of ALL patients and normal to achatinin ( et al, ). of and patients and normal with achatinin for and the of cell by the The after the of cell cells with achatinin against different of achatinin to the of achatinin or The represent the of To the in the on of ALL patients at different of their treatment ( et al, ; Mandal et al, ). In A the which increased by and in C and In after relapse the to a similar to that in A ( Fig the significantly between A and between and C and also between and normal also represents an between the and the peripheral cells the of peripheral being in by in C and and after relapse As normal a of a expression of that the an with expression of 9-O-AcSGs and can as a of et al, in the of normal and ALL patients under different of clinical and the between of achatinin and cells ( et al, ). A C years, and ( et al, ). with achatinin and ( Mandal et al, 1997 ; et al, ). of treatment and normal and cells in in different of treatment ( et al, 1999a ). from ALL patients and normal and to the of et . The of 9-O-AcSGs on the of ALL patients in different of treatment by the quantification of The of in patients of which to in in C and in with the for normal ( Fig However, after relapse the increased to similar to a of of childhood ALL is to patients in clinical may to which as the of disease and ( 1995). of residual in patients in is defined as minimal residual disease It is to to the information on MRD and for further information the is referred to the and ( et al, 1997 ; et al, 1997; et al, 1997 ; et al, et al, ). available for detection of MRD include and techniques. Among the available methods for MRD detection in analysis of and of antigen have as the most and ( & both having a of in ( et al, 1998 ). In both these the use of quantification and analysis of is to false This may after in the identified during the of the disease ( et al, et al, 1998 ), further an and its for clinical This may be by application of both ( et al, ). However, a for the clinical application of these MRD is of analysis can be but the not to analysis ( et al, ). Therefore, an urgent need exists to identify cellular and specific to altered expression could be used for and monitoring of MRD in childhood As the in ( Fig significantly with the disease a study in which patients under different of treatment followed for the as an to the clinical of ALL patients ( et al, ), the for all patients from to and reflected a increase in expression of After the increased to during and during therapy This a increase in and of This could be either to a expression of namely of the and Mandal, or to the of these residues as a of after the to a in and of is is that the could relapse even the the of ( et al, ). of two ALL patients in the above study ( et al, ) are shown in Fig both patients a to with to increased to The in that the in a of which increased further to at and In the a in the from to at which further to at the with Taken together, the of this that the could in this at least Therefore, that this can be used for monitoring of ALL patients as it shows a potential for detection of A of of achatinin of two of an study ( et al, ). is in the after As risk is of in the of for with leukaemia ( & detection of MRD in the of treatment to the development of study that monitoring of using this which can detect with a of in may be used to MRD and risk in the of that patients with may be as patients and are for disease patients having a may be as This may to in The is specific as achatinin binds to and the of ALL their with the of normal The specificity of this is further by the that it shows minimal in patients with and The and of the have been in a study ( et al, the natural of Achatina fulica the of achatinin H, with its can be for to at the an for evaluation of of ALL patients at different of the detection of residual leukaemia is to be used in clinical the analysis should be as frequently as and the should be and to treatment to be to the of The for detection of MRD has its clinical the of our clinical in followed ALL patients in patients at that to normal with to and increased in patients Mandal, to be is the of the to detect of MRD that can be with a in The use of two features of cells could minimize the likelihood of false negative that tandem application of the to detect the specific 9-O-AcSGs with the to detect serum would in the of this as a novel in the of ALL However, still need to these two methods with techniques used for monitoring of and The of sialoglycoconjugates, their structural and their peripheral position on the oligosaccharide of glycoconjugates well to as molecular of various biological sialylation on of ALL patients reported by et in the to of cell-surface negative Subsequently, modification of cell-surface residues to be not but could be with increased potential ( & and cell of their ( Keppler et al, ). As glycobiologists the their with currently available analytical tools to detect sialic acid and its the will from sialic acid as being for to being of have that the linkage of 9-O-AcSA derivatives to the galactose residue for the oligosaccharide chain ( et al, ) and that this can be by antibodies against 9-O-AcSA which are of normal serum ( Zeng et al, 1992 ). Similarly, the 9-O-AcSGs have been as differentiation in processes ( Varki, against sialidases ( et al, 1992 , and of binding and of to murine ( et al, 1992 ). is an important phenomenon in biological from and to an and to the presence of metastasis ( et al, ). a a in and of cellular ( et al, 1995 ). is a present namely sialic acid, the being an structural ( Kelm et al, et al, 1994 ). human 9-O-AcSGs are known to the binding of to its sialic acid ( et al, 1994 ). It has been that 9-O-AcSGs a regulatory to interactions with In this it would be interesting to study whether a similar scenario exists in ALL in which the presence of 9-O-AcSGs has been reported ( Mandal et al, 1997 ; et al, 1999a ). that this increased presence of 9-O-AcSGs on may the of cellular interactions by their binding would of cellular namely cell and and leading to a of function and Taken together, it appears that 9-O-AcSGs may well be an by to sialic acids are known to the of the thereby of the pathway ( that the at C-8 and C-9 of sialic acid are for this function has been in murine of by a O-acetyl group in their increased to ( & . The of 9-O-AcSA in pathway has been in murine cells ( Shi et al, have reported that the of 9-O-AcSGs with differentiation is with an to pathway This that of the group at C-9 by an O-acetyl group In of patients with visceral an increased presence of 9-O-AcSGs has been reported ( et al, 1998 these to to Mandal, As the of ALL patients have increased 9-O-AcSGs ( Sen et al, 1994 ), the of and its with should be Similarly, antibodies against O-AcSGs identified in ALL patients ( Pal et 2000 ), its in has not been The study of biological systems information that may be into development of to disease. information biologically their structural analysis and the characterization of their binding should it to study their in disease conditions and their biological As O-AcSGs and antibodies against O-AcSGs altered expression in the will be to this information and its expression as an to the disease in the of a major in ALL This study by the of and of our clinical of of for of clinical for this our Pal and for their in of and of and Schauer for their and of a from the of and of