Domain organization of the extracellular region of CD45

Antony Symons1, Antony C. Willis2 and A.Neil Barclay3

MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE and 2 Medical Research Council Immunochemistry Unit, Biochemistry Department, University of Oxford, Oxford, OX1 3QU, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CD45 is a large, heavily glycosylated, transmembrane protein phosphotyrosine phosphatase found on all nucleated cells of haematopoietic origin. In lymphocytes, the cytoplasmic phosphatase is necessary for efficient signalling through the antigen receptor but in contrast little is known about the interactions of the extracellular region of the molecule. This consists of a mucin-like region, a novel cysteine-containing region and a region containing three putative fibronectin type III domains. To confirm this organization and to identify parts potentially important for function, we have expressed fragments of the extracellular domain of rat CD45 as recombinant soluble proteins. Proteins corresponding to two, three and four domains of CD45 were expressed in secreted forms. Single domains and constructs for proteins with truncations of the predicted domains were not expressed. This is consistent with the proposed structural organization. Determination of the positions of the disulphide bonds in the N-terminal cysteine-containing region and the first fibronectin type III domain identified novel disulphide bonds within the fibronectin type III domain and an unusual inter-domain disulphide linkage. Circular dichroism spectroscopy indicated that this region of rat CD45 has mainly ß-strand secondary structure and no {alpha}-helical content. These studies support the proposed domain organization of CD45.

Keywords: CD45/cell surface proteins/domain/fibronectin type 3/phosphatase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The transmembrane protein tyrosine phosphatase CD45, is a family of large heavily glycosylated cell surface molecules expressed on all nucleated cells of haematopoietic origin (Thomas, 1989Go; Trowbridge and Thomas, 1994Go). Different isoforms of CD45 are generated by alternative splicing of three exons near the N-terminus of the extracellular region (Streuli et al., 1987Go) and these isoforms range in molecular weight from ~180 to 240 kDa (Barclay et al., 1987Go). Expression of different CD45 isoforms is regulated during haematopoietic cell development and on different cell lineages (Thomas, 1989Go; Trowbridge and Thomas, 1994Go). Notably, functional sub-populations of CD4+ T cells can be defined by expression of the CD45 molecules isoforms (Fowell and Mason, 1993Go; Powrie and Mason, 1990Go). The cytoplasmic region of CD45 contains tandem protein phospho-tyrosine phosphatase domains (Charbonneau et al., 1988Go) which from the study of CD45-deficient cell lines have been shown to be essential for efficient lymphocyte activation through the antigen receptor (Pingel and Thomas, 1989Go; Koretzky et al., 1990Go; Justement et al., 1991Go; Weaver et al., 1992Go). Evidence suggests that CD45 phosphatase activity is required to dephosphorylate and activate the Src-family kinases lck and fyn, important in antigen receptor induced signalling (Mustelin et al., 1989Go, 1992Go; Shiroo et al., 1992Go; Stone et al., 1997Go).

The mechanism by which the CD45 extracellular region affects the phosphotyrosine phosphatase activity is unknown and the role of the different CD45 isoforms in T cell activation is unclear. It has been suggested that specific isoforms differentially affect signal transduction (Novak et al., 1994Go; Okumura and Thomas, 1995Go). However, ligands specific for different CD45 isoforms have yet to be identified. The segregation of cell surface molecules on the basis of the size of their extracellular regions provides an alternative explanation for the differential affect on signalling of different CD45 isoforms (Davis and van der Merwe, 1996Go; Shaw and Dustin, 1997Go). Electron microscopy has shown that expression of all three alternatively spliced exons doubles the size of the CD45 extracellular region from 28 nm for CD45R0 to 53 nm for CD45RABC (McCall et al., 1992Go). The three-dimensional structure of the extracellular region of CD45 is therefore of importance in addressing these issues.

Protein sequence analysis suggests the structure of CD45 resembles that of other transmembrane protein tyrosine phosphatases (Brady-Kalnay and Tonks, 1995Go) whose extracellular parts often contain several fibronectin type III (fn3) domains and immunoglobulin (Ig) like domains. No domain types were noted in the original analysis of the CD45 sequence but subsequently a single and then more recently three fn3 domains were predicted from sequence analysis (Bork and Doolittle, 1993Go; Okumura et al., 1996Go). Fn3 domains consist of ß-strands that form two anti-parallel ß-sheets, similar to the structure of the Ig fold (Leahy et al., 1992Go; Main et al., 1992Go). The proposal that CD45 contains three fn3 domains is supported by the observation that the boundaries of the putative domains also correspond with intron/exon boundaries (Johnson et al., 1989Go). The overall domain structure appears to be conserved between mammals, chickens and horned shark, but the sequence identity between the extracellular regions is very low (~20% identical residues) (Okumura et al., 1996Go). In Figure 1Go, the putative CD45 fn3 domains are aligned with known fn3 domains. These amino acid sequence alignments demonstrate that key residues important for the structural fold of the fn3 domain—for example, alternating hydrophobic residues in the ß-strands that form the hydrophobic core of the protein—are present in CD45 from different species. However, the domains are relatively atypical, for example in containing significant numbers of disulphide bonds which are rare in fn3 domains. Overall the extracellular part of CD45 is predicted to contain a mucin-like region (which includes the regions coded by alternatively spliced exons), one small cysteine rich domain (d1) and three fn3 domains (d2, d3 and d4).



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Fig. 1. Alignment of the putative domains of the extracellular region of rat CD45 with known examples of the fibronectin type III (fn3) domain. Amino acid sequences were aligned using the PILEUP program (Devereux et al., 1984Go). Residues that are identical or conservative substitutions in four or more of the seven sequences aligned are boxed. Positions equivalent to the exon boundaries determined in mouse CD45 are indicated by a double line between residues. The solid bar above the sequence indicates the approximate position of the ß-strands deduced from the structures of other fn3 domains. Sequences are from the SWISSPROT database with accession number and residues for each domain given in brackets; rat CD45 (P04157; d1, 246–339; d2, 339–432; d3, 433–524); human fibronectin precursor (P02751; fn3/8, 1265–1357; fn3/10, 1447–1541); human tenascin precursor (P24821; fn3/3, 801–891); human growth hormone receptor precursor (P10912, 148–252).

 
The atomic structure of the extracellular part of CD45 is required for a molecular understanding of the interactions of CD45. Linear arrays of several domains are difficult to crystallize, however, expression of single or pairs of domains is often effective (Barlow and Campbell, 1994Go; Campbell and Downing, 1994Go). Thus we have expressed fragments of the extracellular region of rat CD45 to confirm the domain organization of the extracellular region and to identify recombinant proteins for NMR spectroscopic and/or X-ray crystallographic structural analysis and as reagents for studying interactions.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fragments of the extracellular region of rat CD45 was expressed using the transient expression vector pEFBOS (Mizushima and Nagata, 1990Go) which was engineered to include six histidines (His-tag) at its C-terminus for purification on Ni-NTA Sepharose. DNA encoding the second putative fn3 domain of the extracellular region of rat CD45 was amplified by PCR using the vector pLC-29 as a template (Barclay et al., 1987Go). SalI and BamHI restriction sites were introduced at the 5' and 3' ends respectively. The 3' primer also included sequence encoding the His-tag and a stop codon, with a BstEII restriction site between the His-tag and CD45 homologous region. The PCR product was digested with SalI and BamHI and ligated into SalI/BamHI cut pEFBOS, previously engineered to include the rat CD4 leader (CD4L) upstream of the SalI site. Further DNA fragments encoding domains of the extracellular region of rat CD45 were amplified by PCR using pLC-29 as a template and combinations of primers with a SalI site in the 5' primer and a BstEII site in the 3' primer. Primers were designed according to the published sequence of rat CD45R0. Each PCR product was digested with SalI and BstEII and inserted, in frame with the CD4 leader and His-tag, into the pEFBOS vector. All constructs were sequenced using the Applied Biosystems Model 373A DNA Sequencer. DNA was transfected into COS-7 cells by the DEAE-dextran method and supernatants harvested after 3–4 days. Expression of recombinant His-tagged CD45 proteins was assayed by precipitation with Ni-NTA Sepharose. Spent medium (1 ml) was incubated with 50 µl 20% Ni-NTA Sepharose for 1 h at 4°C, washed five times with PBS/azide (3000 r.p.m., 1 min) and analysed by SDS–PAGE followed by western blotting with an anti-rat CD45 polyclonal antisera (Sunderland et al., 1979Go).

For each immunoprecipitation, 50 µl 20% Protein G Sepharose suspension was incubated with 1 ml mAb spent tissue culture supernatant (TCS) for 1 h at 4°C, and then washed (3000 r.p.m., 1 min) three times with PBS/azide. A 50 µl aliquot of 20% antibody-coated Sepharose beads were incubated with 1 ml transfected COS-7 cell supernatant for 2 h at 4°C, washed twice with PBS/azide, 50 mM NaCl and a further three times with PBS/azide. Finally, 30 µl loading buffer was added and the samples boiled for SDS–PAGE analysis and western blotting.

Stable expression of the membrane-proximal two domains of rat CD45; DNA encoding the CD4 leader and the membrane-proximal two domains (d1–d2) of rat CD45 with no histidine tag was excised from pEFBOS with XbaI and BamHI and ligated into XbaI/BclI digested pEE14. The expression construct was transfected into CHO-K1 cells and methionine sulfoximine-resistant clones selected as previously described (Davis et al., 1990Go). CD45 d1–d2 expression was quantified using an inhibition ELISA assay. Recombinant rat CD45RO extracellular domain (McCall et al., 1992Go) was adsorbed onto vinyl microtiter plates and the ability of samples to inhibit the binding of a rabbit anti-rat CD45 polyclonal antisera (Sunderland et al., 1979Go) to the plate was followed with an alkaline phosphatase-conjugated mouse anti-rabbit Ig (Sigma Chemical Co.). For large scale production the highest expressing cell line was grown to confluence in cell factories prior to the addition of 2 mM sodium butyrate (Davis et al., 1990Go). Cultures were then left for a further 3–4 weeks prior to harvesting. Soluble d1–d2 CD45 was purified from spent TCS by affinity chromatography using an OX1 antibody affinity column, elution with 50 mM diethylamine–HCl pH 11.5, followed by gel filtration on a Superdex 75 HR10/30 FPLC column (Pharmacia Biotechnology, Herts, UK).

Amino-terminal protein sequencing was carried out using an Applied Biosystems Procise 494A protein sequencer (Perkin-Elmer Ltd, UK). Purified d1–d2 CD45 protein (1 mg) was deglycosylated by incubation with 130 mU PNGaseF (New England Biolabs, Hitchin, UK) at 37°C in 50 mM sodium phosphate pH 7.5 for 48 h. A further 130 mU PNGaseF was added after 24 h. To map disulphide bonds between cysteine residues, 100 µg deglycosylated rat d1–d2 CD45 protein was digested with 4 µg sequencing grade, modified trypsin (Promega) in 100 mM Tris–HCl pH 8.0, 50 mM iodoacetamide for 16 h at 37°C. The digest was then run on an Applied Biosystems 172A microbore HPLC system (Perkin-Elmer Ltd, UK) using an Atlantis C5 reverse phase column (Phenomonex, UK) equilibrated in 0.1% trifluoroacetic acid, 2% acetonitrile and peptides eluted with linear gradients of 2–40% acetonitrile over 60 min followed by 40–100% acetonitrile over 20 min at a flow rate of 150 µl/min. All major peaks were sequenced. Additional disulphides were identified by digestion of 50 µg deglycosylated rat CD45 d1–d2 in 100 mM Tris–HCl pH 8.0, 50 mM iodoacetamide with 1 µg {alpha}-chymotrypsin (Boehringer, sequencing grade) for 6 h at 37°C. Peptides were separated using a Jupiter C18 reverse phase column (Phenomonex, UK) equilibrated in 0.1% trifluoroacetic acid, 2% acetonitrile and peptides eluted with linear gradients of 2–40% acetonitrile over 55 min followed by 40–100% acetonitrile over 15 min at a flow rate of 50 µl/min. All major peaks were sequenced as above.

Circular dichroism spectroscopy measurements were made with a JASCO J-270 spectropolarimeter, using a quartz cuvette with a path length of 1 mm with protein samples in H20 at several concentrations in the range 0.1–0.5 mg/ml. Purified sperm whale myoglobin was obtained from Sigma and monoclonal antibody OX1 was purified from ascites on a HiTrap Protein G affinity column, eluting with 0.1 M glycine–HCl, pH 2.5. The protein concentration of OX1 and CD45 d1–d2 were measured spectrophotometrically using a Coomassie protein assay (Pierce Chemical Co., UK). Spectra were recorded at 25°C between 185 and 260 nm (0.2 nm steps, 20 nm/min and 1 s time constant). Five spectra were averaged for each sample and the spectrum for the buffer alone subtracted.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transient expression of fragments of the extracellular domain of rat CD45

To identify which parts of the extracellular region of CD45 could be expressed as independent recombinant molecules, constructs for individual domains, and combinations of domains, were cloned into a transient expression vector to provide soluble fusion proteins with a C-terminal six histidine tag. COS-7 cells were transfected and the supernatants screened for secretion of histidine-tagged CD45 proteins by precipitation with Ni-NTA Sepharose and western blotting with a rabbit polyclonal antiserum raised against rat CD45. Proteins consisting of two, three or four domains of rat CD45 were expressed in a secreted form. The four domain protein (d1–d4) was expressed as a 70–90 kDa protein consistent with its expected molecular weight (Figure 2Go). The heterogeneity in size is likely to be a result of variation in glycosylation. The membrane-distal pair of domains (d1–d2) is also blotted as a smear at around 46 kDa (Figure 2Go). Eight of the 12 potential N-linked glycosylation sites in the rat extracellular region are in this region of the molecule. The membrane-proximal two domains (d3–d4) and membrane-distal three domains (d1–d3) were expressed in the supernatant (38 and 67 kDa, respectively). However, both appeared to form dimers as a second band was blotted under non-reducing conditions at approximately twice the molecular weight of the lower band (Figure 2Go). The three domains nearest the membrane (d2–d4) were also expressed at a low level (Figure 2Go). In contrast to the expression of multiple domains as soluble proteins, no single domains were expressed in isolation (Figure 2Go). Eight single domain constructs were made and four of these focused on the second putative fn3 domain (d2) which has most similarity to known fn3 superfamily members (23). Extending constructs for fn3 domains from titin at the N- and C-termini beyond predicted domain boundaries, has been shown to affect their levels of expression and stability of the domains (Pfuhl et al., 1997Go). However, extending CD45 domain 2 at N- and C-termini had no affect.



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Fig. 2. Western blot analysis of recombinant proteins consisting of one to four domains of rat CD45 purified by Ni-NTA Sepharose from transiently transfected COS-7 cells. Recombinant proteins of two or more modules were expressed in a secreted form; however, individual modules were not expressed. Two constructs truncated at their N-terminus, indicated by * (see also Figure 3Go), were not expressed.

 
Some insight into the boundaries of each domain was gained by attempting to express proteins with boundaries different from those predicted. Whilst the d1–d2 protein was expressed at high levels, a protein truncated at the N-terminus of d1 by eight residues was no longer expressed (Figure 2Go). Similarly, removal of eight amino acids at the N-terminus of the first putative fn3 domain in the d3–d4 protein lead to loss of expression (Figure 2Go). A summary of the results of expression from all the constructs made is illustrated in Figure 3Go.



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Fig. 3. Schematic representation of the results of expressing fragments of the extracellular region of rat CD45 as recombinant proteins. The extracellular region of rat CD45 is represented in the middle of the figure. Recombinant proteins that were expressed in a soluble form are aligned above, proteins that were not expressed are aligned below. The amino acid sequence at the N- and C-termini of each protein are indicated above and below the whole extracellular region, respectively. The transmembrane domain is coloured black and the mucin region is shaded.

 
Four CD45 mAb bind to recombinant CD45d1–2 and d1–4

Recombinant CD45 proteins that were expressed in transfected COS-7 supernatants were used in immunoprecipitation experiments to localize the binding sites of the four available anti-rat CD45 mAbs; OX1, OX29, OX30 and NDS58. Figure 4Go shows that all the mAbs immunoprecipitated the d1–d4, and d1–d2 CD45 proteins but did not bind to the d3–d4 fragment. This demonstrates that the epitopes for OX1, OX29, OX30 and NDS58 anti-rat CD45 mAbs map to the two membrane-distal domains of the extracellular region and suggests that recombinant d1–d2 CD45 is folded as in the native protein.



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Fig. 4. CD45 mAbs bind to the membrane-distal two domains of CD45. Each anti-rat CD45 mAb (OX1, OX29, OX30 and NDS58) was used to precipitate full length (four domains) CD45 (b), the membrane-proximal two domains (c) and the membrane-distal two domains (d). OX68 mAb was used as a negative control. The ability to precipitate recombinant CD45 was tested by western blot. A control supernatant (a) lacking recombinant CD45 proteins, gave no bands in the positions corresponding to CD45 but a high Mr non-specific band was present in all samples.

 
Stable expression of the membrane-distal two domains of rat CD45

Of the recombinant CD45 proteins that were transiently expressed the membrane-distal two domain protein (d1–d2) was of particular interest for further analysis, because it contained a novel cysteine-rich region of 70 amino acids, and a putative fn3 domain but with low sequence identity to members of this superfamily. The first two domains of rat CD45 (d1–d2) were expressed in CHO cells using the glutamine synthetase expression system as described in Materials and methods. Recombinant CD45 d1–d2 was purified from spent medium from a high expressing cell line by immunoaffinity chromatography followed by gel filtration. SDS–PAGE showed that the purified protein migrated as a 43 to 70 kDa glycoprotein (Figure 5aGo). The yield of d1–d2 CD45 protein was 10–15 mg/l TCS as measured by inhibition ELISA.



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Fig. 5. SDS–PAGE analysis of purified recombinant d1–d2 CD45 protein. (a) The purified protein migrates as a smear with molecular weight from 43 to 67 kDa under reducing (R) and non-reducing (NR) conditions. (b) Deglycosylation with PNGaseF results in a decrease in molecular weight from 43–67 kDa to two major forms at 21 and 23 kDa.

 
Deglycosylation of the membrane-distal pair of domains of rat CD45

The membrane-distal two domains of rat CD45 are heavily glycosylated with eight N-linked glycosylation sites. Peptide N-glycosidase F (PNGaseF) treatment reduced the molecular weight from 43–70 kDa to two major species of approximately 20 and 23 kDa (Figure 5bGo) and a smear from 40 to 30 kDa suggesting deglycosylation was incomplete. The 20 kDa molecular weight form is consistent with complete removal of carbohydrate to the 19.6 kDa peptide backbone but the basis of the 23 kDa band was not obvious and this heterogeneity is not ideal for structural studies. The recombinant d1–d2 protein remained soluble once its N-linked oligosaccharides had been removed as demonstrated by gel filtration (data not shown).

N-terminal sequencing of recombinant d1–d2 CD45 protein

The presence of two forms of CD45 d1–d2, 20 and 23 kDa, after PNGaseF treatment indicated heterogeneity in addition to N-linked glycosylation. N-terminal sequencing of the 20 kDa protein showed that the protein had an extra two amino acids derived from the leader sequence (Figure 6Go) which had been cleaved two residues upstream of that found in CD4 or other proteins where this CD4 leader has been used. This resulted in a short stretch of protein containing serine, threonine and proline residues, characteristic of O-linked glycosylation sites. The 23 kDa protein had the same N-terminus but had been post-translationally modified by O-glycosylation as indicated by the lack of amino acids detected at positions 2 and 4 (data not shown). The presence of two O-linked sugars in the 23 kDa form is compatible with the differences in Mr of the two forms.





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Fig. 6. The membrane-distal two domains of CD45 contain a novel inter-domain disulphide bond. Deglycosylated d1–d2 CD45 protein was digested with trypsin and chymotrypsin, the peptide fragments separated by HPLC and the peptides in all major peaks sequenced. (a) and (b) show the separation of peptides generated from digestion of d1–d2 CD45 with trypsin and chymotrypsin, respectively. (c) The table shows the amino acid sequence of peptides in the major peaks and the yield of each peptide. In peaks containing more than one peptide, disulphide linked peptides are present at equimolar yields. The peptides were completely sequenced and all tryptic peptides ended in K or R (pools A–E) and all chymotryptic peptides end in W, F, Y, K or L (pools F–H). Deglycosylation converts asparagine residues to aspartic acids and this is indicated by `D(N)'. Cysteines involved in disulphide bonds will not be seen in peptide sequencing. `-' indicates positions where residues were undetected. (d) Schematic representation of the disulphide bonds in the membrane-distal two domains of CD45 determined by this analysis.

 
Disulphide mapping of the membrane-distal pair of domains of rat CD45

The two membrane-distal domains of rat CD45 contain 10 cysteine residues, with five cysteines in each putative domain. This implies that either the domain predictions are inaccurate, or each domain has at least one free cysteine, or that there is a disulphide bond between the two domains. Determination of the disulphides is a test of the prediction that d2 has a fn3 fold. PNGaseF-treated d1–d2 protein was digested with trypsin or chymotrypsin, peptide fragments separated by HPLC and all the major peaks were sequenced. The analysis showed that the first two cysteines in the novel cysteine-containing domain (Cys10 and Cys42) form disulphide bonds with the last (Cys69) and middle (Cys47) cysteines, respectively, in the same domain (Figure 6Go). The disulphide bond between Cys10 and Cys69 was observed in five tryptic peptide pools, including peptide pools A and B shown in Figure 6Go. The difference in mobility on HPLC is due to partial cleavage at some sites. The bond between Cys42 and Cys47 was observed in chymotryptic peptide pool F. The first two cysteines in the second putative domain (Cys91 and Cys102) were shown to be linked (tryptic peptide pool E and chymotryptic peptide pool H). Similarly, the last two cysteines in this putative fn3 domain (Cys124 and Cys152) formed a disulphide bond. From the tryptic digest, peptide pool D contained equimolar amounts of Cys124 and Cys152 in two separate peptides (Figure 6Go). Finally, an interdomain linkage between the two domains was observed. The disulphide bond between Cys57 and Cys113 was seen in tryptic peptide pool C, which contained equimolar amounts of these cysteine residues in separate peptides (Figure 6Go).

These results are consistent with the proposed domain structure of CD45 but indicate an unusual interdomain disulphide bond. Modelling the amino acid sequence of domain 2 on the structure of the tenth fn3 module of tenascin (Leahy et al., 1992Go) showed that Cys124 and Cys152 are in close enough proximity to form a disulphide bond between the F and C ß-strands. The bond between Cys91 and Cys102 would link the A and B ß-strands. In the model Cys113 is in the BC loop at the top of the fn3 domain and adjacent to the novel domain. This position is consistent with formation of an interdomain disulphide bond with Cys57 in the novel domain.

Circular dichroism of the membrane-distal pair of domains of rat CD45

To gain some insight into the secondary structure of the two membrane-distal domains of rat CD45, the CD spectra of d1–d2 was measured between 185 and 260 nm and compared with CD spectra of proteins with known structures (Figure 7Go). The spectrum of myoglobin, a predominantly {alpha}-helical protein (Takano, 1977Go), has characteristically intense negative bands at 222 and 208 nm, and a positive band at 192 nm. In contrast, IgG contains only ß-sheet (Harris et al., 1998Go) and its CD spectrum is very different. It has a weak negative band at about 214 nm and a positive peak at 202 nm. The measured CD spectrum of recombinant d1–d2 CD45 was similar to that of IgG with a negative band at about 212 nm and a positive band at ~198 nm. The distinguishing band at 208 nm was not present in this spectrum implying the two membrane-distal domains of rat CD45 have no {alpha}-helical content and probably a high content of ß-sheet structure.



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Fig. 7. Circular dichroism secondary structure analysis of the membrane-distal two CD45 domains indicates a predominantly ß-sheet content and no {alpha}-helical structure. Circular dichroism spectra for purified immunoglobulin (monoclonal antibody OX1 at 0.25 mg/ml), myoglobin (0.13 mg/ml) and d1–d2 CD45 (0.5 mg/ml), were measured from 185 to 260 nm. Spectra are plotted in mean molar ellipticity per residue ({theta} MR) units (deg.cm2.mol–1). The inset to the figure shows the symbols used to plot each protein spectrum.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of combinations of putative domains (Figures 2 and 3GoGo) together with the failure to express truncations of these, provide good evidence for the presence of the three predicted fn3 domains plus a fourth smaller domain with no sequence similarity to any known domain. Proteins corresponding to individual domains were not expressed as soluble proteins suggesting stabilizing interactions with adjacent regions of the molecule (Figure 3Go). This is not atypical for multi-domain proteins and was found in studies on the expression of CD4 domains (Traunecker et al., 1989Go). Thus the pattern of expression of recombinant CD45 proteins (Figure 3Go) is consistent with the domain organization predicted from the primary sequences of mammalian, avian and shark CD45 extracellular region (Okumura et al., 1996Go), namely an N-terminal region with many potential O-linked glycosylation sites, a novel cysteine-containing domain and a region containing three fibronectin type III (fn3) domains. Fn3 domains consist of anti-parallel ß-sheets structurally similar to the Ig fold but with no sequence similarity (Barclay et al., 1997Go). For each fn3 domain in CD45 conserved alternating hydrophobic residues similar to those found in other fn3 domains and forming potential ß-strands can be identified (Figure 1Go). Whilst the overall domain organization appears to be conserved between species, the sequence identity between mammals, chicken and shark is actually very low with only ~20% identical residues and about 40% between rodents and man (Okumura et al., 1996Go). The latter is close to the value for CD8{alpha} and many other cell surface antigens are around 50% identical, including CD2 and CD4 (Barclay et al., 1997Go). It is possible that a large proportion of the polypeptide is not involved in interactions per se but is important in maintaining the general topology of the protein. The finding that all four available mAbs recognized the membrane distal 2 domains and not the membrane proximal region may indicate that the latter is less accessible to mAbs at the cell surface.

Disulphide bond analysis of the membrane-distal two domains of rat CD45 further supports the proposed structural organization. In the rat there are 10 cysteine residues in this part of the molecule with five in each putative domain (Barclay et al., 1987Go). The domain structure proposed here implies there are disulphide bonds in each fn3 domain whereas fn3 domains usually lack disulphide bonds (Figure 1Go). Examples of disulphide-containing fn3 domains are found in cytokine receptors such as gp130 (Hibi et al., 1990Go) and CDw131 (Hayashida et al., 1990Go) and other examples include the Drosophila protein neuroglian (Huber et al., 1994Go) and the neuron and lymphocyte cell surface adhesion molecule L1 (Miura et al., 1991Go). Immunoprecipitation of the CD45 d1–d2 protein with mAbs which recognize native rat CD45 indicated that this protein was correctly folded (Figure 4Go) and the disulphides identified are representative of the normal protein. Modelling of the putative fn3 domain (d2) indicated the cysteine residues within this domain are close enough in space to form bonds. These unusual bonds may be an important feature of CD45 stabilizing the structure of the fn3 domains. The data show that Cys113 in this fn3 domain (d2) forms an inter-domain disulphide bond with Cys57 in d1 (Figure 6Go). In the model, Cys116 is in the BC loop at the top of the fn3 domain in a position that would be adjacent to d1. Disulphide bonds between adjacent domains in a linear array of domains are uncommon. To our knowledge the only other documented example of such a disulphide bond is for myelin associated glycoprotein (Pedraza et al., 1990Go) and it is also likely to be present in other members of this family of sialic acid-binding Ig-like lectin proteins now called `siglec' (Crocker et al., 1996Go). Formation of a disulphide bond between these domains would restrict their orientation relative to one another which may be important for function. Interestingly, the cysteine residues involved in the inter-domain disulphide bond identified here in rat CD45 are conserved in the shark sequence although the number and position of cysteine residues is only approximately conserved between vertebrate species. Chicken and shark CD45 have a significantly different distribution of cysteines compared with each other and to mammalian CD45 species (Fang et al., 1994Go; Okumura et al., 1996Go). The greatest variation between chicken, shark and mammalian sequences occurs in the novel domain. This region is shorter in the chicken; ~48 amino acids shorter than the rat and containing only one cysteine residue (Fang et al., 1994Go) and the chicken protein is likely to have a different structure than other species in this region. However, based on sequence analysis, the three fn3 domains in CD45 appear to be conserved in these species.

An alternative organization of the extracellular region of CD45 based on the chicken homologue has been proposed that includes a spectrin-like domain in place of d1 and d2 (Fang et al., 1994Go). However, protein sequence analysis indicates the similarity to fn3 domains is much higher (Okumura et al., 1996Go). The CD spectrum of the membrane-distal two domains of rat CD45 (d1–d2) presented here (Figure 7Go), clearly shows that the rat CD45 protein contains no {alpha}-helical structure discounting the possibility of a spectrin-like domain in this part of the molecule.

Recent models of T cell activation, involving the segregation of molecules within a contact zone, have been based upon the size of the extracellular domains of the different cell surface proteins known to be involved (Davis and van der Merwe, 1996Go; Shaw and Dustin, 1997Go). The T cell receptor–MHC complex, CD28/CD80 signalling molecules and CD2/CD48 ligand pair are predicted to all have similar dimensions of approximately 15 nm (Davis and van der Merwe, 1996Go; Garboczi et al., 1996Go; Garcia et al., 1996Go) and are thought to aggregate in a contact zone between the T cell and the antigen-presenting cell, with the lipid bilayers closely aligned (Dustin et al., 1998Go). Larger molecules involved in activation, such as integrins and CD45, are excluded from this area probably because of steric constraints (Monks et al., 1998Go). In this model, exclusion of CD45 is essential for the phosphorylation cascade associated with the T cell receptor. The size of CD45 is based on the measurement of the extracellular region made from electron microscopy data. Electron microscopy has shown that the common core of the extracellular region of rat CD45 has a rod-like structure and the polypeptide region containing O-linked glycosylation sites has an extended conformation (McCall et al., 1992Go). The use of the variable exons changes the overall size of the CD45 extracellular domain from 28 nm for the lowest molecular weight isoform (CD45R0) to 51 nm for the largest (CD45RABC) (McCall et al., 1992Go). By comparison, the crystal structure of a fragment of human fibronectin encompassing four fn3 repeats has been determined (Leahy et al., 1996Go) and the structure reveals a rod-like molecule with a long axis of 14 nm. The extracellular region of the mucin-like, leukocyte cell surface antigen, CD43, has been analysed by electron microscopy and its 240 amino acids were shown to have an extended conformation with an overall size of 45 nm (Cyster et al., 1991Go). Based on these measurements the extracellular domain of CD45R0 would be expected to have a long axis of approximately 20 nm [14 + (45x30/240)]. This suggests that the dimensions may have been overestimated from the electron microscopy measurements. The discrepancy of 8 nm cannot be explained by the novel cysteine-containing domain having an unusually extended conformation as gel filtration of the deglycosylated d1–d2 protein showed the molecule to elute at the expected position. A CD45R0 extracellular region approximately 20 nm in length is still significantly greater than the 15 nm distance between T cell and APC lipid bilayers in a contact zone. In addition the larger isoforms formed by alternative splicing of mucin-like regions will be even longer. The large amount of carbohydrate attached to the CD45 protein is likely to keep the extracellular domain orientated away from the lipid bilayer. It should be noted that CD45 in addition to being very large is also very abundant (Barclay et al., 1997Go). Thus, segregation of CD45 from the TCR–MHC–peptide complex due to steric constraints represents a plausible mechanism for T cell activation and it seems likely that the extracellular domain of CD45 interacts with other proteins but with a low affinity. The role of the larger isoforms may simply involve a steric effect due to the increased size leading to CD45 being further excluded from the contact zone making the phosphatase activity more remote from the activation site.


    Acknowledgments
 
We are grateful to Peer Bork for valuable discussions on CD45 sequences and to Shigekazu Nagata for the pEFBOS vector. This work was supported by the Medical Research Council, Arthritis Research Council and the European Union biotechnology programme.


    Notes
 
1 Present address: The R.W.Johnson Pharmaceutical Research Institute, 3535 General Atomics Center, La Jolla, California, USA Back

3 To whom correspondence should be addressed; email: barclay{at}molbiol.ox.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 17, 1999; accepted July 6, 1999.