Cronograma de actividades

The existence of a novel extrinsic attachment was suggested through research into bacterial PI- PLC enzymes. PI-PLC enzymes were amongst the first specific phospholipases identified, through their involvement in the disease, anthrax. An unanticipated side-effect of anthrax was a significantly raised concentration of alkaline phosphatase (ALP) activity in the blood stream, a phenomenon called alkaline phosphatasemia (Smith, 1955).

Investigation of this phosphatasemia showed that it could be reproduced by injection of a

soluble factor derived from Bacillus anthracis (Slein & Logan, 1960). A crude toxin

preparation was injected intravenously into rabbits, and blood was drawn at intervals. The blood samples were analysed for a variety o f serum components, including glucose, glycoproteins, cholesterol, alkaline phosphatase and serum enzymes. As little as 4 hours after injection of the toxin preparation, ALP levels had risen as much as 4 fold, compared with normal levels. The activity decreased slowly, but was still significantly higher than normal 71

hours after injection. Similar effects were observed after intravenous injection o f the B.

anthracis toxin into rats, mice and guinea pigs.

This toxic effect was also observed after intravenous injection of a culture filtrate derived from

Bacillus cereus, inducing a marked increase in the blood ALP levels (Slein & Logan, 1962). Fractionation techniques, using N,N’-diethyl-aminoethyl cellulose, were designed to separate the phospholipase enzymes from the culture filtrate. The resultant fractionate contained two phospholipase enzymes, one of which displayed the capacity to induce alkaline phosphatasemia, which was termed phosphatasemia factor (PF), while the other enzyme inhibited the phosphatasemia. Intravenous injection of relatively large amounts o f the purified PF resulted in the depletion of bone ALP (Slein & Logan. 1963).

Originally, this enzyme was termed lecthiniase, because of the ability o f lecithin to inhibit its activity. The lecithinase was classified as a phospholipase, through its ability to cleave both phosphoryl choline, phosphoryl ethanolamine and phosphoryl inositol from the phosphatase group in a number of substrates. Upon closer examination, however, it was revealed that the PF enzyme specifically hydrolysed phosphatidyl inositol (PI), but had no activity against phosphatidyl Choline (PC), sphingomyelin (SM) or phosphatidyl ethanolamine (PE) (Slein & Logan, 1965). It was, therefore, suggested that ALP was attached to the membrane via a phosphatidylinositol (PI) structure (Izekawa, 1976), and that the target of the enzyme was this PI. The enzyme was, therefore, re-classified as phosphatidylinositol-phospholipase C, and represented a phospholipase with specificity for the PI structure. PI-PLC enzymes have been

identified in other bacterial species, including Staphylococcus aureus (Low & Finean, 1977

(b)) and Bacillus thuringiensis (Taguchi et a l, 1980)

The association between PI and ALP could either exist as a non-covalent binding, between a specific binding site on the enzyme molecule and the PI molecule, or as a covalent linkage. The suggestion of a non-covalent link was dismissed, largely due to two observations. Firstly, non-covalent interactions can be disrupted, by exposure to high ionic strength, divalent cations or extremes of pH. None of these disruptions promoted the release of significant amounts of ALP. In addition, non-covalent linkage is also inconsistent with the observation that the free- ALP, released after exposure to PI-PLC, could not re-associate with membranes (Low & Zilversmit, 1980). The evidence, therefore, argued against a non-covalent binding of the ALP to membrane-located PI. Evidence that the link was covalent, however, required analysis of the mechanism by which the phospholipase enzymes dissociated Pl-linked substrates.

3.4 GPI anchored proteins, as substrates of PI-PLC

In vitro treatment o f membranes with bacterial PI-PLC resulted in the release of various

membrane proteins, including 5’-nucleotidase (Taguchi & Ikezawa, 1978) and

acetylcholinesterase (AChE) (Low & Finean, 1977(b)). Analysis of these proteins, however, showed that they contained a structure that was different from the PI structure. The evidence to support the theory that an alternative structure existed was difficult to obtain, as experimental studies had specifically removed the protein from its anchoring structure. In order to determine the nature of the anchor, it was essential to identify a wide range of substrates in which this novel structure was thought to occur, and to purify these proteins without the removal of the anchor. This was achieved through the identification o f the substrates. Thy-1 and mfVSG, whose characterisation will be discussed more fully in the subsequent Sections.

3.5 Structural analysis of glycoproteins

3.5.1 Thy-1 glycoproteins

Thy-1 is a cell surface glycoprotein, of relative molecular mass 18,000 daltons, one third of

which represents carbohydrate (Morrison et a l, 1984). Thy-1 is a major constituent of

thymocytes and neurons (Reif & Allen, 1966). The rat protein was selected for study because it is present in high quantities in cells, and because large quantities of rat thymus and brain could be obtained. The protein sequences o f both rat and mouse Thy-1 have been analysed, and the mature protein consists of 111 to 112 amino acids, respectively (Williams & Gagnon, 1982).

However, Thy-1 was susceptible to cleavage from the cell surface by S. aureus PI-PLC enzyme,

which supported the idea that the protein contained a PLC-sensitive segment, in addition to the mature protein sequence (Boothroyd, 1985).

Initial analysis of the protein sequence indicated that Thy-1 did not contain a typical transmembrane sequence. However, examination of the cDNA of Thy-1 revealed a sequence of 31 codons that were not present in the mature protein sequence. These 31 amino acids contained an extremely hydrophobic stretch of 20 amino acids, including six leucine residues,

although it was not believed that this stretch represented a transmembrane anchor (Seki et a l,

1985 (a)). The conclusion was drawn that Thy-1 is created as a molecule o f 142 amino acids, 31 o f which are removed, during the transformation into the mature product. However, pulse chase experiments, designed to study the transformation o f the immature protein to the mature protein, found no evidence of post-translational processing. It was concluded that these 31 nucleotides provided a signal sequence, which directed the immature protein to the biosynthetic machinery responsible for attaching the non-protein tail, with the con-current loss o f the 31

nucleotides (Seki e t a l, 1985 (b)).

Extraction of the intact Thy-1 protein was possible as a consequence of the ability of Thy-1 to bind to non-ionic detergents. The C-terminal of Thy-1 protein was extracted using Brij 96 detergent, with purification through a Biogel column. However, the protein could not be analysed by standard gas chromatography analysis because of the presence o f the detergent in the Thy-1 extract. This problem was bypassed by the development of a thin layer analysis method (Williams & Tse, 1985). Results from this technique indicated that, in addition to the known Thy-1 protein, mannose, myo-inositol, glucosamine, galactosamine, phosphate, glycerol and stearic acid were detected.

More detailed information concerning the anchor of Thy-1 was provided through the specific

release o f the protein from the fatty acid chains using 6". aureus-dQnwQd PI-PLC (Homans et a l,

1988), after which the anchor was subjected to one and two dimensional NMR, gas chromatography mass spectroscopy and exoglycosidase digestion. The structural information that was obtained as a result o f this analysis is discussed in Section 3.7.

3.5.2 Variable Surface Glycoprotein from Trypanosoma Bruceii

The study o f the novel anchor was further advanced through research into the surface coating of Trypanosoma bruceii, the parasite responsible for the cattle disease, nagana. The related

parasites, T. rhodesiense and T. gambiense, cause sleeping sickness in humans (Borst &

Rudenko, 1994). Variable Surface Glycoprotein (VSG) proteins form a dense coating on the surface o f each individual parasite. The ability of trypanosomes to periodically shed and replace this coating with a different sub-type of VSG, a process called antigenic variation, is

an important factor in the ability of trypanosomes to evade the host’s immune system. The process by which antigenic variation was accomplished seemed a novel area of research.

VSG, derived from T bruceii, was chosen an ideal candidate for study. The parasite is

relatively easy to cultivate in large quantities, and the VSG can be purified to homogeneity. Two forms of VSG, membrane-form (mf) and soluble (s), were specifically extracted from the

surface o f parasite (Cardoso de Almeida et a l, 1983), and analysis o f the anchor bearing

mfVSG was, therefore, possible.

3.6 Chemical analysis of the membrane attachment of VSG

Structural analysis of the membrane-form (mf)VSG indicated that it contained unusual chemical components, including a glycan, containing mannose, glucosamine and variable amounts of galactose (Holder & Cross, 1981) and an ethanolamine-amide, linked to the C- terminal amino acid of the target protein (Holder, 1983). The anchor was termed the Glycosyl- Phosphatidyl-Inositol (GPI) anchor, with the proteins being termed GPI-anchored proteins

(Ferguson e t a l , 1985).

Following the detailed analysis of Thy-1 and mfVSG, a number of other GPI-anchored proteins

were identified. These included alkaline phosphatase (ALP) (Malik and Low, 1986),

membrane-form bovine erythrocyte acetylcholinesterase (mfAChE) (Hoener et a l, 1990),

decay accelerating factor (DAF) and basic fibroblast growth factor (bFGF) (Metz et a l, 1994).

These substrates were extracted, purified and characterised.

3.7 Structure of the GPI-anchor

The complete structure of the GPI-anchor was first determined using the VSG substrate

(Ferguson et a l, 1988) after which time the mammalian Thy-1 antigen was analysed (Homans

et a l, 1988). A number of analytical techniques have been employed, including gas chromatography, mass spectroscopy, specific chemical cleavages and sequential digests with

exoglycosidases, chromatography and NMR spectroscopy (Ferguson et a l, 1988). Essentially

the same backbone structure was identified in both proteins. Subsequently, the composition and arrangement of the chemical constituents of the GPI-anchor has accumulated from many different structure studies. The GPI anchor contains a number of components, which are linked to one another in a precise sequential arrangement, and is schematically illustrated in Figure 3.1.