engineering proteins that bind to cell surface carbohydrates

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E L S E V I E R Clinica Chimica Acta 266 (1997) 13-22

Engineering proteins that bind to cell surface carbohydrates

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J. Pau l L u z t o ' , J. Mark B r y a n t , Peter W. Taylor b

aDepartment of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, UK

bResearch Centre, Novartis Pharmaceuticals, Wimblehurst Road, Horsham, West Sussex RH12 4AB, UK

Received 1 May 1997; received in revised form 22 July 1997; accepted 23 July 1997

Abstract

Carbohydrate residues covalently linked to plasma membrane proteins and lipids often provide specific markers at the cell surface. Traditionally such carbohydrate structures have been identified using antibodies and lectins. However problems of affinity and lack of specificity have restricted their usefulness. Protein engineering offers a way round these difficulties. In the case of some specialised cell surface carbohydrate structures, such as polysialic acid, enzymes may be useful analytical tools. Endosialidases specific for polysialic acid have recently been cloned and sequenced. © 1997 Elsevier Science B.V.

Keywords: Carbohydrate residues; Cell markers; Endosialidase

1. I n t r o d u c t i o n

Cell surface glycoproteins and glycolipids are often very specific markers of individual cell types or cells at a particular stage of development [1]. The carbohydrate moieties on these molecules have a wide range of known biological functions including cell-cell interaction [2], although there are many examples where the function remains unclear [3,4]• Specific oligosaccharide

Corresponding author.

0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0009-8981 (97)00162-9

14 J.P. Luzio et al. / Clinica Chimica Acta 266 (1997) 13-22

sequences are also important targets for recognition by pathogenic toxins and micro-organisms. There is a long history of identifying carbohydrate structures on cells and proteins using both anti-carbohydrate antibodies and lectins (naturally occurring proteins which bind carbohydrates; [5]), particularly in blood typing. In recent years, enzymes have played an increasingly, important role in characterizing carbohydrate structures. In the case of polysialic acid, a long chain polymer of ot-2,8-1inked sialic acid residues found on the surface of some pathogenic bacteria as well as specific mammalian cells, specific bacteriophage endosialidases have proven at least as important as antibodies in molecular characterization [6].

A problem with many of the reagents used to study cell surface carbohydrate structures has been the low affinity of binding. In some cases antibodies have shown no detectable binding to carbohydrate ligands under conventional binding assay conditions. Increasing avidity by presenting oligosaccharides in a clustered state has overcome this problem [7]. A further difficulty in the case of lectins has been the lack of mono-specificity [8]. Recent work using the techniques of protein engineering offers the prospect of overcoming these limitations, leading to new uses of proteins that bind to carbohydrate structures on the cell surface.

2. Antibodies

Polyclonal antisera are the end product of a complex series of events in the immune system which may include the suppression of lymphocyte clones producing antibodies to some parts of the immunogen. In contrast the production of a monoclonal antibody effectively freezes and immortalizes a stage in the development of an antibody-producing clone. Thus in addition to the ability to generate essentially unlimited amounts of a monospecific reagent, monoclonal antibody technology allows the production of antibodies to unexpected parts of antigenic molecules not represented in a polyclonal antiserum. The advent of monoclonal antibodies, with their built in specificity, was important in identifying a wide range of carbohydrate structures found on the cell surface. They were particularly important in the detection of differentiation antigens i.e. antigens which change during cell differentiation and maturation, many of which were found to be carbohydrates [1]. They also found widespread use in diagnostic pathology, but were not without problems, in particular due to low affinity or unexpected cross reactions. The ability to engineer antibodies genetically has provided ways around these difficulties and offers new opportunities in diagnostic pathology [9].

Using the polymerase chain reaction, cDNA clones for immunoglobulin molecules, or fragments of them, can now be easily obtained from hybridoma cells producing monoclonal antibodies. Protein engineering may be used to alter

J.P. Luzio et al. / Clinica Chimica Acta 266 (1997) 13-22 15

the binding characteristics of a particular antibody [10] and the availability of phage display techniques to select recombinant antibody fragments allows the relatively simple selection of antibodies of improved affinity and specificity [ 11 ]. In a study of bacterially expressed Fab and single chain variable domain (scFv) preparations of an antibody specific for the Salmonella serogroup O-polysaccharide, low affinities due to rapid dissociation were observed [12]. Mutant scFv molecules with enhanced binding properties were selected and the enhanced binding shown to be due primarily on their propensity to form dimers. It was suggested that higher affinity might be engineered into anti-carbohydrate antibodies by manipulating dissociation rate constants.

In addition to a role in diagnosis, recombinant antibodies also offer new therapeutic possibilities. In particular there is much current interest in the delivery of recombinant immunotoxins to tumour cells. The immunotoxin is engineered as a hybrid protein made up of an antibody fragment directed at an appropriate cell surface marker and a toxic protein capable of killing the cell to which the immunotoxin has bound [13,14].

2.1. Lectins

Plant lectins have been widely used to characterize variations in carbohydrate structures found on cell surface glycoproteins and glycolipids [15]. They have also been used, sometimes in combination with other methods, to distinguish isoenzymes that differ only in carbohydrate content [16]. Legume lectins have been amongst the most useful and several proteins from this family have been crystallised and their structure determined. Although the number of subunits in these lectins varies, the individual subunits all have a jelly-roll tertiary structure consisting of a flat six-stranded [3-sheet and a curved seven-stranded [3-sheet interconnected by loops of various lengths [17]. Sequence hypervariability in the loops which constitute the binding site is the basis of the wide range of carbohydrate specificities. The availability of crystal structures allows the possibility of rational site-directed mutagenesis to alter specificity and/or affinity.

In the case of one lectin, peanut lectin (PNA), which has been widely used in the detection of Thomsen-Friedenreich antigen (Gal[31,3GalNac) on the sur- faces of malignant cells and immature thymocytes, mutagenesis has resulted in a recombinant lectin of improved specificity [18]. Thomsen-Friedenreich antigen is a chemically well-defined tumour associated antigen of non-oncofoetal origin with a proven link to malignancy in man. PNA also recognises N-acetyllactosamine (Gall31,4GlcNAc) which is present at the termini of several cell surface glycoproteins. The crystal structure of PNA-lactose revealed, in addition to the expected interactions with the residues constituting the binding site, the presence of a leucine (L212) at a position close enough to bind the acetamido

16 J.P, Luzio et al. I Clinica Chimica Acta 266 (1997) 13-22

group on N-acetyllactosamine. Two mutants, L212N and L212A were con- structed and their carbohydrate specificities determined. The L212A mutant showed increased affinity for N-acetyllactosamine. Thus, the replacement of leucine by an amino acid with a shorter aliphatic chain (alanine) facilitated the binding of N-acetyllactosamine. In contrast, the asparagine mutant (L212N) did not bind N-acetyllactosamine presumably because substitution of leucine by a polar residue of equivalent size (asparagine) compromised the binding of N-acetyllactosamine by disrupting the favourable interaction between the acetamido group and the hydrophobic leucine side chain. The L212N mutant is thus a more specific reagent than its wild type counterpart.

2.2. Endosialidases and polysialic acid

Polysialic acid (PSA), is a linear carbohydrate polymer of residues of N-acetyl neuraminic acid (NeuSAc) joined by ot-2,8-ketosidic linkages ( [6], [19]; Fig. 1 a). PSA occurs in a diverse, yet restricted, set of biological situations but in all cases the chains are a covalent modification of cell surface glycoconjugates. A number of pathogenic strains of bacteria, including Escherichia coli K1 and Neisseria meningitidis Group B, express PSA as part of the bacterial capsule. In higher organisms, PSA is found in the envelopes of certain fish eggs, in Drosophila embryos, as a post-translational modification of the neural cell adhesion molecule (NCAM) in mammalian neurons and the developing embryo, and as an oncodevelopmental marker in particular tumour types. In Escherichia coli K1 and Neisseria meningitidis Group B, PSA may exceed 200 residues in length and is a key virulence determinant thought to mediate resistance to phagocytosis and complement mediated killing. In mammals PSA, often exceeding 50 residues in length, is added post-translationally to N-linked glycan chains on its principle cartier, NCAM, by a polysialyltransferase which is probably resident in the transGolgi network [20]. The addition of PSA to NCAM is developmentally regulated in the central and peripheral nervous systems where its expression modulates NCAM mediated adhesion [21]. In this context, PSA expression is implicated in influencing a number of important events, including neurite development, axon guidance, embryogenesis and the retention of cell plasticity in neuronal tissue. In the adult mammal PSA-NCAM is restricted to defined regions of the central nervous system. In addition it is also expressed on the surface of a variety of tumour cell types (Table 1) and is correlated with the ability to metastasise. Thus, small cell lung carcinoma cell lines exhibit a decreased propensity to aggregate in vitro and an increased ability to metastasise in vivo when they have PSA on the cell surface [22].

Two types of reagent, antibodies and endosialidases, have been essential in the detection of PSA. Although both polyclonal antisera and some PSA-specific monoclonal antibodies have been reported, the very poor immunogenicity of

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Table 1 Human tumour types and cell lines expressing PSA

Tumours and cell lines of neuroendocrine origin

Medullary thyroid carcinomas Medulloblastomas Neuroblastomas Phaeochromocytomas Pituitary adenomas Small cell lung carcinomas Schwannomas

Tumours and cell lines of non-neuroendocrine origin

Rhabdomyosarcomas Accute lymphoblastic leukemias of pre-B cells Wilms tumours (nephroblastomas) Teratomas (immature and intermediate) MCF7 breast cancer cells

In the majority of cases, the presence of PSA was identified using anti-PSA antibodies. The PSA is found attached to NCAM in all instances except in the MCF7 cell line, where the identity of the protein is not known. References to many of these tumours are found in Troy et al. [6] and to MCF7 cells in Martersteck et al. [30].

PSA has made it difficult to prepare them. PSA-specific endosialidases have been purified from E s c h e r i c h i a c o l i K1 specific bacteriophages. These bacteriophages were originally isolated to aid in the clinical identification of E s c h e r i c h i a c o l i K1 infections which can result in high mortality rates in cases of neonatal meningitis. Bacteriophage K1E endosialidase is thought to be the protein responsible for initial binding of the bacteriophage to host bacteria by specifically recognising and hydrolysing the PSA. It has been proposed that K1E endosialidase could be used in the diagnosis and therapy of K1 meningitis, septicaemia or bacteraemia due to the enzyme's high specificity for hydrolysing a-2,8-sialosyl linkages [23]. The primary nucleotide sequence of the bacteriophage K1E endosialidase gene contains an open reading frame predicted to encode a 90 kDa polypeptide chain [24]. The major protein sequence features (Fig. lb) are two copies of an eight amino acid sialidase motif found in numerous bacterial and viral exosialidases and a sequence matching the eight amino acid consensus 'P-loop' frequently found in proteins that bind nucleotides and also some carbohydrate binding proteins (Table 2). The central region of the molecule exhibits a high level of sequence identity with bacteriophage K1F endosialidase [25], although the latter is considerably extended at the carboxy- terminus. A closely related endosialidase E sequence has also been reported [26]. The function of the eight amino acid sialidase motifs is unknown. Although they were suggested to be necessary for sialidase function [27], crystal

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structures suggest a structural role. Several bacterial and viral exosialidases have been shown to have a six-bladed 13-propeller structure in which each blade is a four-stranded anti-parallel [3-sheet. In the exosialidase from Salmonella typhimurium, the sialidase motifs form the turn between the third and fourth strands of each sheet on the periphery of the propeller with an aromatic group tucking in between adjacent sheets [28]. Mutagenesis studies on the sialidase motifs of Clostridium perfringens exosialidase, have provided data consistent with them playing a role in the tertiary structure of the enzyme [29].

The only published reports of efforts to express recombinant endosialidase suggest considerable difficulties in this process. Attempts to express endosialidase F as a fusion protein with an amino-terminal extension were unsuccessful [25]. Although expression of endosialidase E with a carboxy-terminal extension was reported [26], activity was assessed by the ability to reduce the antigenicity of PSA rather than by a conventional enzyme assay. In addition the expression vector used is unlikely to result in the ability to recover large amounts of fusion protein since the mature enzyme appears to be proteolytically processed at the carboxy-terminus such that the 90 kDa primary translation product is converted to a 76 kDa subunit in the mature enzyme [24].

3. Concluding remarks

At present there are relatively few examples of engineered proteins that bind to specific cell surface carbohydrates. However, these few examples demonstrate the possibilities that exist to improve the specificity and affinity of both antibodies and lectins for diagnostic and therapeutic purposes. In the case of enzymes, present opportunities include the prospect of identifying unusual sources of enzymes specific for particular carbohydrate structures and preparing these enzymes as recombinant proteins to provide reliable sources of diagnostic reagents.

Acknowledgements

JMB held a Medical Research Council collaborative studentship. Experimen- tal work on bacteriophage E endosialidase was funded by Ciba Pharmaceuticals (now Novartis Pharmaceuticals), Horsham, UK.

J.P. Luzio et al. / Clinica Chimica Acta 266 (1997) 13-22 21

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engineering proteins that bind to cell surface carbohydrates

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