Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents

A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody.     

Alpha-helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly cross-linked beta beta tropomyosin

Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked beta beta tropomyosin two-chain coiled coils. Cross-linking was performed by reaction of sulfhydryls with either ferricyanide or 5,5'-dithiobis(2-nitrobenzoate) (NbS2). The extent of reaction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either by titration of residual sulfhydryls with NbS2 (ferricyanide cross-linking) or by determination of mixed disulfide (protein-S-SbN) through reaction with dithiothreitol (NbS2 cross-linking). The results indicate approximately 90% conversion to molecules with interchain cross-links at both C-36 and C-190. Thermal unfolding curves are compared with those obtained previously for non-cross-linked species. The curves are indistinguishable up to approximately 40 degrees C. Above approximately 40 degrees C, the doubly cross-linked species is more stable, but the transition is less steep. This relationship is also compared with that found between alpha alpha tropomyosin (a similar coiled coil made of a genetic variant chain having a sulfhydryl only at C-190) and its singly cross-linked derivative. Thermal curves for alpha alpha and beta beta non-cross-linked species are very similar, alpha alpha being somewhat more stable. For cross-linked alpha alpha, however, the curve sags at temperatures somewhat below the region of principal cooperative loss of helix, the latter occurring at higher temperature but with the same steepness as in the non-cross-linked case. The sag has been ascribed to a "pretransition" in the region of C-190. Thus, doubly and singly cross-linked species differ in that the former show no pretransition and decreased steepness in the principal transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

大肠杆菌L-酒石酸脱氢酶b亚基体外分子交联

通过PCR技术扩增大肠杆菌L-酒石酸脱氢酶b亚基(L-tartrate dehydratase beta subunit, TtdB)野生型与Cys/Ser突变型目的基因, 构建带6×His标签的诱导型表达载体pTrcHisC-TtdB。重组蛋白以包含体形式存在, 应用TALON固定化金属亲和树脂(Immobilized metal affinity chromatography, IMAC)以变性的方法纯化, 通过分步透析逐步去除变性剂的方法复性, 复性率可达70%。将复性后的两种蛋白通过热诱导去折叠和氧化重折叠方法进行体外蛋白质分子交联实验。SDS-PAGE分析表明: 野生型TtdB在其变性的临界温度反应时, 出现交联二聚体和多聚体; 在氧化重折叠后SDS-PAGE前加入100 mmol/L DTT时, 交联强度明显减弱。这种DTT打不开的交联即为异肽键交联; 若在其氧化重折叠反应液中加入DTT则没有任何交联。突变型TtdB在与野生型TtdB相同的热诱导去折叠条件下, 完全没有二聚体和多聚体的形成。     

Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [≈2 biotins/ IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [¹²⁵I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200–300 kDa range. By contrast, direct mixing with [¹²⁵I]streptavidin rapidly gave larger oligomers of ⪢669 and ≈440–669 kDa molecular mass, respectively. The biodistribution of the 200–300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440–669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.     

Synthesis of "long", hydrophilic, protein-cross-linking reagents

Since most of the protein cross-linking reagents in use are strongly hydrophobic, their length cannot be increased beyond approximately 20 Å between the protein-reactive groups, before denaturation of most proteins becomes noticeable at already a very few cross-links per molecule. The synthesis of longer reagents, coupling to lysine or cysteine side chains, and containing strongly hydrophilic oligoproline chains, is described. As they bear an azodye, linking the oligoproline parts, the cross-links effected are amenable to a mild cleavage by reduction with dithionite. A trifunctional reagent was constructed by reacting trimesinic acid chloride with β-alanine ethyl ester; the carboxyl groups of this amino acid could then be activated for protein cross-linking by reactions leading to the hydrazide, and azides.To compare the new reagents with the compounds in use at present, they were tested out on hemoglobin. The amount of reagent molecules coupled to the protein, and the fractions bifunctionally attached, as well as interchain linking were determined. The “long” reagents reached a distinctly higher efficiency in interchain cross-linking in this system, while showing smaller denaturing effects upon the protein. Thus, more than 11 reagent residues could be coupled to the hemoglobin tetramers without changes in its spectrum indicating denaturation of the heme environment, while shorter and more hydrophobic reagents had permitted the attachment of not more than four to six crosslinks.     

Selective inhibition of high- but not low-affinity interleukin 2 binding by lectins and anti-interleukin 2 receptor alpha antibody

The present study demonstrated that various reagents can specifically reduce the affinity of high-affinity interleukin 2 receptor (IL-2R) but not that of low-affinity IL-2R. They included lectins such as wheat germ agglutinin (WGA), concanavalin A and phytohemagglutinin, and a chemical cross-linker, glutaraldehyde, in addition to anti-IL-2R monoclonal antibodies, HIEI and H-47. The affinity of the high-affinity IL-2R was reduced when the cells were treated with WGA or H-47 before, but not after, addition of 125I-labeled interleukin 2 (IL-2). Their inhibitory effects were also demonstrated by the chemical cross-linking method. On treatment with the reagents, the IL-2 binding to both IL-2R alpha and beta chains was inhibited and these inhibitory effects were seen only when the reagents were added before IL-2 addition, like their high-affinity reducing effects. These results support a supposition that the high affinity IL-2R is generated by assembly of the alpha and beta chains, and suggest that the IL-2 binding to IL-2R alpha and beta chains could induce stable constitution of the high-affinity state of IL-2R, but these affinity modulating reagents could perturb the optimum association between alpha and beta chains to generate the high-affinity IL-2R.     

Effects of interchain disulfide cross-links on the trypsin cleavage pattern and conformation of myosin subfragment 2

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diabetes-associated mutations in insulin: consecutive residues in the B chain contact distinct domains of the insulin receptor

How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe(B25) projects from the surface of insulin, whereas Phe(B24) packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe(B24) at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the alpha subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 beta-helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular alpha subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase.     

Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine.

Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl.3,3'-dithiobispropionimidate*2HCl (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M(w)) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the N/P ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intra-cross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.     

Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6.

Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degradation by a variety of chemical denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), beta-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50 degrees C). DTT and beta-mercaptoethanol caused more rapid degradation of the sheath than cyanide or sulfite. Treatment of the sheath with 1 N NaOH resulted in rapid breakdown, while treatment with 1 N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7808-7818, 1993) were progressively dissociated into random masses during DTT-induced degradation. Quantitation of disulfide bonds with DTT showed that the sheaths contained approximately 2.2 mumol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 mumol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of [14C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-molecular-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissociated fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20 to 25% protein. These results confirm that the sheath fibrils consist of high molecular-weight heteropolysaccharide-protein complexes. We hypothesize that proteins in the fibril complexes provide interfibril cross-linking to maintain the structural integrity of the sheath.     

Mapping of the carboxyl terminus within the tertiary structure of transducin's alpha subunit using the heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine

A heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine (125-ACTP), has been synthesized. 125I-ACTP has been used to derivative reduced sulfhydryls of the retinal G protein, transducin (Gt), to form a mixed disulfide bond under mild, nondenaturing conditions (pH 7.4, 4 degrees C). The resulting disulfide was easily cleaved using reducing reagents. A 200-fold molar excess of 125I-ACTP relative to Gt resulted in the incorporation of 1-1.3 mol of the 125I-N-(3-iodo-4-azidophenylpropionamido)cysteine moiety of ACTP into Gt alpha. In contrast to 125I-ACTP, dithionitrobenzoate and dithiopyridone derivatized six sulfhydryls in native Gt. Incubation of a 10-fold molar excess of 125I-ACTP relative to Gt resulted in the derivatization of 0.75-0.9 and 0.1 mol of reduced sulfhydryls/mol Gt alpha and beta, respectively. Gt gamma was not derivatized by 125I-ACTP. Thus, Gt alpha was preferentially derivatized by 125I-ACTP. Tryptic digestion and amino acid sequencing of Gt alpha indicated that both Cys-347 near the carboxyl terminus and Cys-210 between the second and third consensus sequences forming the GTP-binding site were derivatized by 125I-ACTP in a ratio of approximately 70 and 30%, respectively. Thus, both Cys-210 and Cys-347 are labeled, even though derivatization by 125I-ACTP does not exceed 1 mol of SH/mol Gt alpha. It appears that derivatization of one sulfhydryl, either Cys-210 or Cys-347, excludes labeling of the second cysteine either by steric hindrance or induced conformational change making the second cysteine inaccessible to 125I-ACTP. Consistent with this finding was the observation that pertussis toxin-catalyzed ADP-ribosylation of Cys-347 inhibited 125I-ACTP derivatization of Cys-210. Derivatization of Gt alpha at either Cys-210 or Cys-347 by 125I-ACTP inhibited rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate binding to Gt, mimicking the effect of ADP-ribosylation of Cys-347 by pertussis toxin. ACTP contains a radioiodinated phenylazide moiety which, upon activation, can cross-link the derivatized cysteine to an adjacent polypeptide domain. Following reduction of the disulfide, the [125I] iodophenyl moiety will be transferred to the azide-inserted polypeptide. When photoactivation of the phenylazide moiety of 125I-ACTP after sulfhydryl derivatization was performed, insertion of the Cys-347 which contains Cys-210, was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.     

Identification of bull protamine disulfides

We have identified the disulfide cross-links in bull protamine by titrating intact bull sperm with dithiothreitol (DTT) and following the modification of each cysteine residue with tritiated iodoacetate. The derivatization of each cysteine was monitored by a combination of HPLC, peptide mapping, and protein sequencing. Analyses of total free sulfhydryls show that all seven of the bull protamine cysteines are cross-linked as disulfides in mature sperm. The first disulfide is reduced at a DTT:protamine cysteine (DTT:Cys) ratio of 0.3 and the last at a ratio of 2.0. Intra- and intermolecular disulfides were identified by correlating the reduction of specific disulfides with the dissociation of protamine from DNA in partially reduced sperm and sperm treated with N,N'-ethylenedimaleimide, a bifunctional disulfide cross-linking agent. Three intermolecular and two intramolecular disulfides were identified. The results of these experiments demonstrate that the amino- and carboxy-terminal ends of the bull protamine molecule are folded inward toward the center of the molecule and are locked in place, each by a single intramolecular disulfide bridge. Three intermolecular disulfides cross-link neighboring protamine molecules around the DNA helix in such a manner that the protamines cannot be dissociated from DNA without first reducing the interprotamine disulfides.     

Covalent cross-links between the gamma subunit (FXYD2) and alpha and beta subunits of Na,K-ATPase: modeling the alpha-gamma interaction

This study describes specific intramolecular covalent cross-linking of the gamma to alpha and gamma to beta subunits of pig kidney Na,K-ATPase and rat gamma to alpha co-expressed in HeLa cells. For this purpose pig gammaa and gammab sequences were determined by cloning and mass spectrometry. Three bifunctional reagents were used: N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), disuccinimidyl tartrate (DST), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide (EDC). NHS-ASA induced alpha-gamma, DST induced alpha-gamma and beta-gamma, and EDC induced primarily beta-gamma cross-links. Specific proteolytic and Fe(2+)-catalyzed cleavages located NHS-ASA- and DST-induced alpha-gamma cross-links on the cytoplasmic surface of the alpha subunit, downstream of His(283) and upstream of Val(440). Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys(347) or Lys(352) in the S4 stalk segment. Mutational analysis of the rat gamma subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys(55) and Lys(56) in the cytoplasmic segment. DST and EDC induced two beta-gamma cross-links, a major one at the extracellular surface within the segment Gly(143)-Ser(302) of the beta subunit and another within Ala(1)-Arg(142). Based on the cross-linking and other data on alpha-gamma proximities, we modeled interactions of the transmembrane alpha-helix and an unstructured cytoplasmic segment SKRLRCGGKKHR of gamma with a homology model of the pig alpha1 subunit. According to the model, the transmembrane segment fits in a groove between M2, M6, and M9, and the cytoplasmic segment interacts with loops L6/7 and L8/9 and stalk S5.     

The assignment of chain specificities for anti-Ia monoclonal antibodies using L cell transfectants

The chain specificities of 18 Ak and 26 Ab-reactive anti-Ia monoclonal antibodies have been determined. L cells were transfected with haplotype-matched (A alpha k:A beta k, A alpha b:A beta k) or haplotype-mismatched (A alpha k:A beta b, A alpha b:A beta k) cDNA pairs, lines expressing high levels of surface A complex were selected, and antibody reactivity with a panel of reagents was assessed by cytofluorimetric analysis. Most of the antibodies recognized a determinant specified by one chain, either alpha or (more commonly) beta. A few examples of more complex determinants were also observed. A knowledge of the chain specificities of anti-Ia monoclonal antibodies should prove useful for a variety of studies aimed at dissecting Ia structure-function relationships.     

Mapping of the mastoparan-binding site on G proteins. Cross-linking of [125I-Tyr3,Cys11]mastoparan to Go

Mastoparan (MP) activates GTP-binding regulatory proteins (G proteins) by promoting GDP/GTP exchange through a mechanism similar to that of G protein-coupled receptors (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186). [Tyr3, Cys11]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [Tyr3,Cys11]MP in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [125I-Tyr3,Cys11]MP bound covalently to the alpha subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys11]MP. Cross-linking was enhanced by the addition of beta gamma subunits, but no cross-linking to beta gamma subunits was observed. Cross-linking was inhibited by incubation of Go with guanosine 5'-O-(thiotriphosphate) and Mg2+ and was reversed by incubation with DTT or 2-mercaptoethanol. Stoichiometry of labeling was consistent with the cross-linking of one molecule of [125I-Tyr3,Cys11]MP/alpha subunit, and CNBr hydrolysis of the [125I-Tyr3,Cys11]MP-alpha o adduct yielded one major labeled peptide fragment of approximately 6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant alpha o showed that cross-linking occurred at Cys3. No alpha o sequence was obtained from the same fragment prepared from bovine brain alpha o, which is blocked by a myristoyl group at Gly2. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal region of G protein alpha subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.     

Interaction of the alpha beta dimers of the insulin-like growth factor I receptor is required for receptor autophosphorylation.

We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.     

Alpha-helix to random coil transitions of two-chain coiled coils: experiments on the thermal denaturation of beta beta tropomyosin cross-linked selectively at C36

Current ideas on unfolding equilibria in two-chain, coiled-coil proteins are examined by studies of a species of beta beta tropomyosin that is sulfhydryl blocked at C190 and disulfide cross-linked at C36 (.beta-beta.). The desired species is produced by a seven-step process: (1) Rabbit skeletal muscle, comprising predominantly alpha alpha and alpha beta species, is oxidized with ferricyanide, cross-linking both species at C190. (2) The product is carbamylated at C36 of beta chains, using cyanate in denaturing medium at pH 6. (3) All C190 cross-links are reduced with dithiothreitol (DTT). (4) All C190 sulfhydryls are permanently blocked by carboxyamidomethylation. (5) Chromatography on carboxymethylcellulose in denaturing medium is used to separate C190-blocked alpha chains from C190-blocked, C36-carbamylated beta chains. (6) The latter are decarbamylated in denaturing medium by raising the pH to 8.0. (7) The C190-blocked beta chains are renatured and cross-linked at C36 by ferricyanide. The procedure and the quality of the final product are judged by NaDodSO4/polyacrylamide gel electrophoresis, titration of free sulfhydryls, and electrophoretic analysis of trypsin digestion products. Thermal unfolding curves are reported for the resulting pure .beta-beta. species and for its DTT-reduction product. The latter (.beta beta.) show equilibrium thermal unfolding curves that are very similar to those of the parent beta beta noncross-linked species. The .beta-beta. cross-linked species unfolds in a single-phase, cooperative transition with a melting temperature intermediate between the pretransition and posttransition shown by its cross-linked counterpart, the C190 cross-linked, C36-blocked species (.beta-beta.), which was studied earlier. These transitions are compared with one another and with that of the doubly cross-linked species, beta-(-)beta, in the light of two extant physical models for such transitions. The all-or-none segments model successfully rationalizes the data qualitatively for the .beta-beta. and .beta-beta. species if the usual postulates of greater inherent stability of the amino vs the carboxyl end of the molecule and of strain at each cross-link are accepted. However, the same model then requires that the beta-(-)beta species be the least stable of the three, whereas experiment shows the opposite, thus falsifying the all-or-none segments model. The continuum-of-states model is also qualitatively in accord with data on the .beta-beta. and .beta-beta. species.(ABSTRACT TRUNCATED AT 400 WORDS)