Carbon-13 NMR study of switch variant anti-dansyl antibodies: antigen binding and domain-domain interactions

A 13C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical VH, VL, and CL domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of these antibodies has been selectively labeled with 13C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by following the procedure described previously for the switch variant antibodies labeled with [1-13C]Met [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 270-278]. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, we discuss how 13C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.     

Structure of a mouse immunoglobulin G that lacks the entire CH1 domain: protein sequencing and small-angle X-ray scattering studies

The structure of a short-chain IgG2a antibody, which is a member of the family of mouse anti-dansyl switch variant antibodies with identical variable regions but different heavy-chain constant regions [Dangl, J.L., Parks, D. R., Oi, V. T., & Herzenberg, L. A. (1982) Cytometry 2, 395-401], is reported. Amino acid sequencing analyses have demonstrated that in the short-chain IgG2a antibody the entire CH1 domain is deleted whereas the hinge region remains intact. Small-angle X-ray scattering data were collected for the short-chain IgG2a antibody and compared with those for the switch variant IgG1, IgG2a, and IgG2b antibodies with the normal heavy chain. It has been concluded that deletion of the CH1 domain results in a large structural change and the short-chain IgG2a antibody possesses an elongated molecular shape with a much smaller hinge angle as compared with the normal IgG2a antibody that is a Y-shaped molecule.     

Lambda cro repressor complex with OR3 DNA: 15N NMR observations

15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing ¹⁵N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a ¹⁵N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.     

Validation of peptide MS/MS spectra using metabolic isotope labeling for spectral matching-based shotgun proteome analysis

We report an isotope labeling shotgun proteome analysis strategy to validate the spectrum-to-sequence assignments generated by using sequence-database searching for the construction of a more reliable MS/MS spectral library. This strategy is demonstrated in the analysis of the E. coli K12 proteome. In the workflow, E. coli cells were cultured in normal and (15)N-enriched media. The differentially labeled proteins from the cell extracts were subjected to trypsin digestion and two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC QTOF MS/MS) analysis. The MS/MS spectra of the two samples were individually searched using Mascot against the E. coli proteome database to generate lists of peptide sequence matches. The two data sets were compared by overlaying the spectra of unlabeled and labeled matches of the same peptide sequence for validation. Two cutoff filters, one based on the number of common fragment ions and another one on the similarity of intensity patterns among the common ions, were developed and applied to the overlaid spectral pairs to reject the low quality or incorrectly assigned spectra. By examining 257,907 and 245,156 spectra acquired from the unlabeled and (15)N-labeled samples, respectively, an experimentally validated MS/MS spectral library of tryptic peptides was constructed for E. coli K12 that consisted of 9,302 unique spectra with unique sequence and charge state, representing 7,763 unique peptide sequences. This E. coli spectral library could be readily expanded, and the overall strategy should be applicable to other organisms. Even with this relatively small library, it was shown that more peptides could be identified with higher confidence using the spectral search method than by sequence-database searching.     

Affinity labeling of residues within Hv2 of guinea pig anti-azobenzenearsonate antibodies of different isotypes and from different strains

Anti-p-azobenzenearsonate (ARS) antibodies of IgG1 and IgG2 isotypes produced in inbred strain 13 and strain 2 guinea pigs were affinity labeled with N-(bromoacetyl)-3-[(p-arsonophenyl)azo]-L-tyrosine (BAAT) or N-(bromoacetyl)-p-arsanilic acid (BAA). BAAT was shown to modify approximately 50% of the binding sites specifically and BAA approximately 30%. Both reagents preferentially modified residues in the heavy (H) chain to the extent that it contained over 80% of the affinity label associated with the native molecule. At least 80% of label borne by the variable domain of the H chain (VH) was found in the second hypervariable region (Hv2). BAAT labeled all anti-ARS antibodies exclusively at position N-59, which contains a lysyl residue. BAA labeled predominantly tyrosine at N-57 and, to a lesser extent, lysine-59 and tyrosine-50. Comparison of Hv2 sequences in anti-ARS and in antibodies reactive with other haptens has shown that tyrosine at N-50 and N-57 as well as lysine at N-59 is distinctive of antibodies with anti-ARS specificity, thus implying their involvement in antigen binding. The predominant sequence of Hv2 was identical in anti-ARS IgG1 and IgG2 molecules induced in either inbred guinea pig strain following either carrier priming or conventional immunization. Although limited variability does occur among the various populations of anti-ARS antibodies in certain residue positions in Hv2, no significant differences in the binding affinities or in the indexes of heterogeneity were seen among the various kinds of anti-ARS antibodies.     

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

Localization of the human complement component C3 binding site on the IgG heavy chain

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.     

1H, 13C, and 15N resonance assignments for a ferrocytochrome c553 heme by multinuclear NMR spectroscopy

A novel strategy has been used to assign the 1H, 13C, and 15N resonances of the heme in Anabaena 7120 ferrocytochrome c553. 13C[13C] double-quantum coherence spectroscopy was used to delineate the heme carbons, 1H[13C] single-bond correlation spectroscopy was used to define the attached protons, and 1H[15N] multiple-bond correlation spectroscopy was used to assign the nitrogens. 1H[13C] multiple-bond correlation spectroscopy confirmed many of the assignments. Proteins were labeled uniformly with 13C or 15N to obtain the required spectral sensitivity.     

Implications of 15N-metabolic labeling for automated peptide identification in Arabidopsis thaliana

We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.     

Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer

The use of uniform 13C,15N labeling in the NMR spectroscopic study of RNA structures hasgreatly facilitated the assignment process in small RNA oligonucleotides. For ribose spinsystem assignments, exploitation of these labels has followed previously developed methodsfor the study of proteins. However, for sequential assignment of the exchangeable andnonexchangeable protons of the nucleotides, it has been necessary to develop a variety of newNMR experiments. Even these are of limited utility in the unambiguous assignment of largerRNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei.These problems can largely be overcome by the additional use of base-type selectively13C,15N-labeled RNA in combination with a judicious use of related RNAs with basesubstitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNAaptamer in complex with AMP. Complete sequential 1H assignments, as well as the majorityof 13C and 15N assignments, were obtained.     

[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13C evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110 kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time period.     

A polar substituent-containing acylation agent for the radioiodination of internalizing monoclonal antibodies: N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate ([131I]SGMIB)

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.     

IgG Subclass and Heavy Chain Domains Contribute to Binding and Protection by mAbs to the Poly γ-D-glutamic Acid Capsular Antigen of Bacillus anthracis

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3→IgG1→IgG2b→IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.     

Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid

Membrane-associated decay accelerating factor (DAF) of human erythrocytes (Ehu) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the Ehu acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact Ehu with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated from urine. Nitrous acid deamination cleavage of Ehu DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) released the [125I]TID label in a parallel fashion as from [125I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [3H]ethanolamine resulted in rapid 3H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. Our findings indicate that DAF and AChE are anchored in Ehu by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi.