Journal of Autoimmunity (1990) 3 (Supplement), 91-96

Structure Function Analysis of In Vitro MutatedCD4 and Major Histocompatibility Complex Class II

Gene Products

Jam.es McCluskey, Lars Kjer-Nielsen and Rozanne Blok

Department of Pathology and Immunology, Monash Medical School, CommercialRoad, Melbourne, Australia 3181

The Class II major histocompatibility molecules are implicated in theinitiation of antigen-driven autoimmune responses by CD4-positive Tcells. In order to study the structure and function of CD4 and MHC Class IImolecules, strategies were developed with the intent ofgenerating secretedforms of these molecules by in vitro mutagenesis of the respective genes. Afull length eDNA encoding an expressible human CD4 molecule was rnuta­genized to introduce a premature stop codon corresponding to residue 367located 8 amino acids amino terminal to the start of the predicted trans­membrane region. Following DNA-mediated gene transfer of the mutantgene, secreted CD4 was detected in the supernatant of transiently trans­fected COS-l cells. Surface expression of the membrane-bound form ofCD4 was detected under the same conditions.

In an attempt to create a secreted form·of the mouse Class II moleculeI_Ak the exons encoding the connecting stalk, transmembrane and cyto­plasmic domains of both the a and Pchains were replaced by the corres­ponding exons from the gene encoding a secreted Class I-like molecule,QlOb. Transfer of these genes into mouse L cells failed to generatedetectable secreted I-A molecules.

In view of the secretion of CD4 reported in other mutagenesis studies, itis concluded that very subtle differences in the structure of the COOH­terminus can influence the folding, solubility or transport of the CD4molecule. In addition, the assembly of heterodimeric Class II moleculesmay require membrane anchorage of the separate chains or some othercontribution from the COOH-terminal domains ofthe a and pchains.

The Ia or Class II molecules of the major histocompatibility complex (MHC) arereceptors which form a binary complex with processed foreign antigen which in turnis recognized by the antigen receptors on T cells [1]. The CD4 molecule on thesurface of T cells probably enhances the interaction between the MHC Class IImolecules and the T-cell receptor by binding a conserved region of the Class IImolecule [2]. Evaluation of the structural basis and functional contribution of these

91

0896-8411/90/020591 +06 $03.00/0 © 1990 Academic Press Limited

92 J. McCluskey et at.

WT MUT

3 ' 5" 3' 5;T

~]i

WT C04 MUT-1 MUT-2~_ ._J ~ THR !

.....- ~--- 1 -...;;;=- - "= A g] A

~ l- =aI - --_ - C TRP C STOP_ ~ _ e; -=-,,-:, i T

-~ - - g]-=' --:=:$ . ---=- , ~ . - :; ..-' - ....,.. G SER

== -.--' ~ -:: = .......

. \ ~- - ~ ]- - - - - THRC T A G C T A G C T A G

39 3 '5" 5 '

Figure 1. Autoradiograph of mutant and wild type CD4 nucleotide sequence. A full length eDNA,encoding human CD4 [9], was cloned into Ml3 mp9 and a synthetic oligonucleotide 5'­CGGGGTGGAICATGTGGGCAG-3' was used to produce a single point mutation (G to Atransition) thus changing amino acid residue 367 from a TRP to a premature stop codon 5' to the predictedtransmembrane coding region [11]. Following mutagenesis two independent mutants (MUT-l andMUT-2) and the wild type gene (WT) were sequenced through the relevant region by the dideoxy chaintermination method, using a synthetic oligonucleotide primer designed to anneal immediately down­stream of the mutated area (5'- TTGGCGCCTTCGGTGCCGGCA-3').

various receptor interactions is confounded by the integral membrane anchorage ofthe molecules involved. Recently, genes encoding secreted forms of mouse Class Imolecules have been engineered by replacing the exons encoding the a3 domain,connecting stalk, transmembrane and cytoplasmic domains of a normal Class I gene(H-2Dd

) with the corresponding exons from the gene encoding a secreted Class I-likemolecule, QlQb [3]. Secreted forms of the CD4 molecule have also been described inwhich premature stop codons placed 5' to the transmembrane exon result in defectivemembrane anchorage of the mutant translated proteins and secretion into themedium by transfected cells [4-8]. The potential utility of these approaches inaddressing receptor ligand interactions led us to attempt to derive secreted forms ofthe mouse Class II molecule I-Ak using the strategy which was successful in pro­ducing secreted Class I molecules. In addition, we independently derived an in uitromutant human CD4 gene with a premature stop codon corresponding to residue367 located 8 amino acids amino-terminal to the predicted transmembrane region.Secreted CD4 protein, but not mouse I-A\ was detectable in the supernatant of therelevant transfected cells.

Results

In order to generate a secreted form of CD4 a cDNA encoding expressible CD4antigen [9] was mutagenized to create a premature stop codon at position 367. Apartfrom the introduced mutation, the DNA sequences obtained (300-400 nucleotides)were identical between wild type and mutant clones, as shown in Figure 1. Becausethe complete mutant CD4 gene was not sequenced following mutagenesis two inde­pendent mutant CD4 clones were studied. Thus wild type and mutant CD4 geneswere subcloned into the expression vector pcEXV-3 [10]. This vector was chosenbecause it works well in providing stable transformant L cells as well as providingamplified gene transcription in transfected COS-l cells. Reconstructed CD4 geneswere then co-transfected with the selectable thymidine kinase (tk) gene into L cellsand COS-I cells and were then stained with the OKT4 monoclonal antibody. Both

CD4 and MHC Class II structure/function studies 93

MUT-1 MUT-2 WT-l

sWT-2

li

5iL

i5

iL

I5

iL

I5

UT

L

KD

10092

69

46

30

1

Figure 2. Irnmunoprecipitated proteins from transfected cells. COS-l cells were transfecred with 10 ugof wild type (WT-1 and WT-2) or mutant CD4 (MDT-1 and MDT-2) DNA" and radiolabeled 48 hlater in medium containing 200 ~Ci/mL "S-methionine. Postnuclear detergent cell lysares (L) or culruresupernatants (S) were reacted with a mixture of the anti-CD4 monoclonal antibodies OKT4 and DAKO­T4 and immunoprecipitates were adsorbed onto protein A sepharose beads, washed and fractionatedunder reducing conditions on a 12.5 0 0 polyacrylamide gel containing SDS. The resultant autoradiographrepresents a 48-h exposure. Precipitates of transfected cells formed using an irrelevant, isorype-matchedmonoclonal antibody (data not shown), and anti-CD4 immunoprecipitates ofuntransfected cells, failed todetect any specific bands following autoradiography.

COS-I and L cells transfected with the wild type CD4 genes revealed bright specificstaining (data not shown). In order to determine whether COS-I cells transfectedwith the mutant CD4 genes were secreting CD4 molecules these cells were radio­labeled with 35S-methionine and then analysed for the presence of immuno­precipitable CD4 by SD S-polyacrylamide gel electrophoresis. As shown in Figure 2,immunoprecipitates formed using a mixture of anti-CD4 antibodies specificallydetected a radiolabeled band with a molecular weight of approximately 48 kDa in theculture supernatant of cells transfected with two independent clones of mutatedCD4. This protein appeared heterogeneous in size suggesting glycosylation of the

94 J. McCluskey et at.

k:- =1-AOl. or~

.... =Ql0b

_ = vector

Class 11=

010

(b )

Figure 3. (a) Construction of hybrid MHC Class II/Class I genes. Subclones of the I-A a and Pchaingenes [13, 14] were prepared to include only the first three exons and part of the third intron of each genesuch that the region encoding the transmembrane and cytoplasmic parts of the molecules was deleted.These subclones were then ligated into a deletion mutant of the QI0b gene which contained part of the 4thintron and remaining 3' sequences of this gene. All constructs were mapped for multiple predictedrestriction endonuclease sites and the Bal31 deletion mutant of the QI0b gene was sequenced to confirmthe presence ofappropriate splicing signals. H, Hind III; Bg, Bgl II; Xh, Xho 1; B, Barn HI; E, EcoRl; S,SaIl; X, Xba 1; L=Leader exon, aI, a2, PI, p2=corresponding exons; TM = Transmembrane exon.Some of the indicated restriction sites were destroyed during the gene construction. (b) Predictedstructure ofhybrid Class II/Class I protein.

mature molecules and consistent with secreted CD4 antigen. The membrane form ofCD4 was detected as a 55 kDa molecule precipitated from cells transfected with thewild type CD4 gene.

The structure of the hybrid I_Ak/QlQbgenes and their expected protein productare shown in Figure 3(a) and 3(b). In order to determine whether expression of thehybrid I-Ak/Ql0b genes resulted in secretion of I-Ak-like dimers these genes wereco-transfected with the tk gene into mouse L cells which were then biosyntheticallyradiolabeled in the presence Of35S-methionine. There was no detectable radiolabeledI-Ak-like protein in the cell supernatant of transfected cells although immuno­precipitable ~2-microglobulinwas readily demonstrable under the same conditions(data not shown).

Discussion

The ability to study ligand-receptor interactions between different cell surfaceglycoproteins is hampered by the many other receptor-ligand interactions which

CD4 and MHC Class II structure/function studies 95

take place between molecules on interacting cells. To simplify analysis of CD4molecular interactions as well as those of the Class II MHC molecule we haveattempted to generate secreted forms ofthese molecules. The gene encoding CD4 wasmutagenized in vitro to introduce a premature stop codon 8 amino acids from thepredicted start of the transmembrane region. Cells transfected with independentclones of the mutant CD4 gene secreted a 48 kDa protein which was specificallyimmunoprecipitated by a mixture of anti-CD4 monoclonal antibodies and revealedsize heterogeneity consistent with a glycosylated protein. Other groups haveattempted to engineer secreted CD4 [4-8]. The introduction of a stop codon atposition 363 [4] failed to produce secreted CD4; however astop codon at the equivalentofposition 370 [5] or at position 371 [6],372 [6, 7], 375 [4] or 378 [4] all resulted in thesecretion of CD4 by transfected cells. From these data there appears to be a criticallength of polypeptide which can be deleted from the region amino terminal to thetransmembrane anchor without disturbing the folding (or transport) of the CD4antigen. CD4 is believed to possess four immunoglobulin-like domains. The apparenteffect ofthe COOH-terminal residues upon the folding ofCD4 mutants may thereforereflect a short connecting stalk between the membrane and globular domains £15].

Strategies for engineering the secretion of MHC Class I proteins have recentlybeen developed and exploit the fact that only the heavy chain of these dimericmolecules is an integral membrane glycoprotein. Thus, exon shuffling of the 0.3,transmembrane and 3' coding sequences between H-2Dd and the gene encoding thesecreted class I-like molecule oro- results in a gene which directs secretion of H-2D d/Q10b chimeric molecules. Because of the success in obtaining secreted hybridClass I/QIOb molecules we derived analogous gene constructs in which the trans­membrane and 3' sequences ofthe I-Ak a.and ~ genes were replaced by the equivalentexons of the QIQb gene. However, transfection of these genes into mouse L cells didnot lead to secretion of detectable I-Ak molecules. Two independent sets of hybridI-Ak/Ql0b genes were constructed, such that the intron sequences at the 5' end ofthe Q10btransmembrane exon were of varying length to minimize the chance ofpre­mRNA splicing errors. In addition, DNA sequencing of this intron demonstratedintact putative lariat branch points and splice acceptor sites (data not shown). Finallyboth the I-A a. and ~ subclones and the QIQb sub clone used for constructing thehybrid genes have been shown to splice correctly in other constructs (unpublishedresults). Thus the most likely explanation for failure to detect secreted I -A moleculesin the transfected cells is that the mutant a.and ~ chains fold or dimerize improperly.Although evidence indicates that the assembly of interallelic pairs of I-A a and Pchains is controlled by residues in the amino-terminal domains [16] this does not ruleout an important role for the membrane-proximal domains and/or other COOH­terminal residues in the overall dimeric assembly of I-A a and ~ chains. Thetransmembrane-coding exons of Class I and II MHC genes also encode the part ofthe protein which links the globular external domains to the transmembrane­spanning domain. This region of Class II a. and ~ chains may playa role in thedimeric assembly of the two chains. Alternatively, tethering of I -A molecules in themembrane may be required for their dimerization.

Note added in proof: Fisher et al. [4] have re-assigned the amino terminus of CD4 tocorrespond to residue +3 in the numbering of Maddon et al. [9]. The amino acid

96 J. McCluskey et at,

numbering used in this study was based upon Maddon et al. [9] and accordinglyshould be deducted by 2.

Acknowledgements

This work was funded by grants from the Australian NH & MRC, AmericanArthritis Foundation, Kellion Diabetes Foundation and a Monash University SpecialGrant. Ms R.B. is recipient of a Monash University Postgraduate Scholarship andMr L.K-N. receives a Commonwealth Postgraduate Scholarship.

References

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15. Classon, B. J., J. Tsagaratos, I. F. C. McKenzie, and I. D. Walker. 1986. Partial primarystructure of the T4 antigens of mouse and sheep: assignment of intrachain disulphidebonds. Proc. Natl. Acad, Sci. (USA) 83: 4499-4503

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