Cation dependency of BGPa m ediated adhesion has been a subject of controversy w ith both Ca^+ dependent (Rojas et al, 1990) and Ca2 + in d ep en d en t (Oikawa et al, 1992) adhesion being rep o rted by two independent groups. In the previous chapter, treatm ent of CHO-BGPc cells w ith 5mM EDTA containing m edium for half an hour, greatly inhibited their capacity for adhesion to the soluble BGP constructs (Chapter III, Fig.3.13). Using the same procedure no cation dependency was found for the CHO-BGPx' cells. A dhesion to the soluble BGP dom ain fusion proteins rem ained the same w ith or w ithout previous treatm ent of the cells with 5mM EDTA (Fig.4.10).
2 0 0 0 0 « 1 5 0 0 0 o c E 1 0 0 0 0 - SI C H O -B G P x ’ E3 No EDTA ü SmMEDTA o u o o Li. u_ u. UL 1 1 1 ■ Z CM CO m < CO T - Û < m Ü <
F ig .4 .1 0 BGPc-Fc [N , N A IB I and N A 1B 1A 2] d o m a in fu sio n p rotein s and CD33-Fc proteins (Chapter II, Fig. 2.2) w ere attached to 96 w e ll plates via a rabbit anti-hum an Ig bridge. CHO-BGPx' transfectants labelled w ith ^ H -T hym idine w ere a llo w e d to bind to these constructs either in the presence of cations or 5m M EDTA. The adherent cells w ere solu b ilised and their radioactive content determ ined b y P scintillation counting. W ells coated w ith BSA and CD33-Fc served as n egative controls for binding. The data sh o w n here are from a sin gle experim ent perform ed in triplicate and are representative of three separate experim ents.
DISCUSSION
The existence of several BGP isoform s, including one consisting of only the N -term inal dom ain followed by the transm em brane an d cytoplasmic dom ains, BGPx and BGPx' for the short cytoplasm ic tail, (Barnett et al. 1993) lead to the idea that it m ight be possible to test the possibility of a N -N interaction for the BGP hom ophilic adhesion. C onstruction of a BGPx' splice variant equivalent w as achieved by using a tw o step PCR based procedure. The resulting cDNA com prises the Leader sequence, N -term inal dom ain, 9 base pairs of the A l dom ain (thought to code for the am ino acids in the hinge betw een th e N an d A l dom ains), the transm em brane and the cytoplasm ic coding sequences. The predicted cDNA w as confirm ed by sequencing w ith no am ino acid substitutions th at m ig h t resu lt from nucleotide m isin co rp o ratio n d u rin g the PCR process being detected. F urtherm ore u p o n electroporation into CHO cells a recognisable protein was being expressed on the CHO cytoplasmic m em brane. A ntibody staining confirm ed th at the protein obtained seem ed to have the correct three dim ensional folding and th at the A l, B1 and A2 dom ains w ere no longer there. A ntibodies know n to react w ith the BGP N -term inal dom ain (C hapter III, Table 3.1) still stained the chim eric p ro tein w hile antibodies against the other BGP extracellular dom ains w ere now negative (Fig.4.4). W estern blot analysis show ed a significant decrease in size for the BGPx' w hen com pared to the BGPc isoform , confirm ing th at the BGPx' isoform was a m uch sm aller protein w ith a m olecular w eight (less than 30 kDa) consistent w ith the deletion of the three IgC2-like constant dom ains present on the BGPa and c isoforms. Confocal analysis of the staining of the new CHO-BGPx' cell line show ed a rem arkable sim ilarity to the patern obtained for the CHO-BGPc cell line w ith the BGPx' m olecule concentrating m ostly on the cell surface and particularly on the sites of cell to cell contact.
In chapter III it has been show n, u sin g the different BGPc dom ain co n stru cts, th a t h o m o p h ilic ad h esio n o f BGPc to th e CHO-BGPc transfected cells requires the N -term inal dom ain of BGPc. Here, using the BGP N-Fc construct and the CHO-BGPx' cell line, confirm ation th at this is a N -N term inal dom ain interaction w as done. The CHO-BGPx' cells w ere able to adhere to the N-Fc construct thus confirm ing th at at least in the initial ad h esio n process no o th er extracellular d o m ain is necessary. Time course experim ents for the N -N term inal interaction show th at
adhesion persists for u p to 3 hours. The dim inished aggregation after 2 hours for BGP isoforms lacking the A2 reported by Rojas et al. (Rojas et al, 1990) does not seem to occur in the experim ental system used here. A small decrease in adhesion was observed after 90 m ins of incubation b u t was follow ed by a new increase that w as m antained u p to the end of the assay.
As obtained for the BGPc isoform, adhesion of the CHO-BGPx' to the N - Fc construct, also seems to be tem perature dependent. H ow ever cation dependency w as not found for the BGPx' isoform. C ation dependency for the BGPa molecule has alw ays been controversial (Rojas et al, 1990; O ikaw a et al, 1992). G enerally the ad h esio n process for m olecules belonging to the im m unglobulin su p er fam ily has been fo u n d to be cation in d ep en d en t. Some scientists have fo u n d th at in aggregation assays and at low levels of expression in transfected cells, BGPs require calcium and physiological tem peratures for aggregation (Rojas et al, 1990; T urbide et al, 1991; O ikaw a et al, 1992) w hereas high levels of BGP expression abrogate the calcium dependency (Obrink, 1991; M cCuaig et al, 1992). In our system, although the level of expression of BGPc on the CHO cells is high, calcium and tem perature dependency still occurs for th e CHO-BGPc ad h esio n to the d ifferen t d o m ain fu sio n p ro tein s. H ow ever w hen the CHO-BGPx' cells w ere used no calcium dependency could be shown. It is possible th at m ore BGPx' then BGPc molecules are expressed at the CHO cell surface. P roduction of the BGPx' isoform seems to be m ore efficient than th at of the BGPc isoform . In fact the sam e pattern w as seen w ith the production of the fusion proteins by the transfected Cos7 cells w ith the N-Fc m olecule being the m ore efficiently produced followed by the NA IBI-Fc m olecule and finally the NA1B1A2- Fc fusion protein. Smaller molecules seem to be easily produced by the transfected cells.
CHAPTER V:
PEPTIDE INHIBITION OF THE BGP N -N TERMINAL
INTERACTION.
INTRODUCTION
In the previous chapter the involvem ent of both N term inal dom ains in the adhesion process betw een tw o BGP proteins w as established. M ost Ig fam ily m em bers have been show n to in teract w ith th eir respective ligands via the N -term inal dom ain. The three dim ensional structure of the N dom ains of som e of these proteins, CD4, REI, CD2, have already been resolved by X-ray crystallography and MEN (Wang et al, 1990; Epp et al, 1974; Driscoll et al,1993 respectively). Three dim ensional com puter m odelling based on sequence sim ilarities to these Ig fam ily m em bers is also available for the CEA molecule (Bates et al, 1992). Based on the data available, w ith the help of Dr. P. Bates at the ICRF, M olecular M odelling Unit, three peptides predicted to contain the am ino acids responsible for the b in d in g properties of the BGP N -term inal dom ain, w ere designed. They span p a rt of N dom ain C C and FG P strands. The CC'FG face on the N dom ains of the Ig fam ily m em bers, w hose th ree dim ensional s tru c tu re have been resolved, is the one th a t is m o st com m only available for interaction w ith other m olecules. It faces o u tw ard s and is not predicted to be covered by carbohydrates. This chapter looks at the ability of these peptides to inhibit adhesion of the CHO-BGPc a n d /o r CHO-BGPx' cell lines to the BGP [N-Fc ] fusion protein. Their capacity to elicit integrin m ediated adhesion is also investigated. Furtherm ore tw o anti-CEA m onoclonal antibodies, HD-11 and AH-11, w ere also tested for their capacity to block BGP hom ophilic adhesion.
RESULTS