The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMobile Fc region in the Zie IgG2 cryoglobulin: comparison of crystals of the F(ab')2 fragment and the intact immunoglobulinK. R. Ely, P. M. Colman, E. E. Abola, A. C. Hess, D. S. Peabody, D. M. Parr, G. E. Connell, C. A. Laschinger, and A. B. EdmundsonCite this: Biochemistry 1978, 17, 5, 820–823Publication Date (Print):March 7, 1978Publication History Published online1 May 2002Published inissue 7 March 1978https://pubs.acs.org/doi/10.1021/bi00598a011https://doi.org/10.1021/bi00598a011research-articleACS PublicationsRequest reuse permissionsArticle Views67Altmetric-Citations45LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Digestion of IgG† Zie with trypsin produced two fragments which differed from the characteristic tFab and tFc normally obtained from tryptic cleavage of IgG. These two unique fragments, designated t 1 and T s were isolated from a tryptic digest of IgG Zie by gel filtration in dissociating solvents. T, was comprised of two intact light chains covalently bound to the shortened γ chains which commenced at His 101‡ and remained predominantly intact to the C-terminus. T s consisted of most of the variable region of the γ chains, probably inclusive of residues 1–100. Cleavage of IgG Zie at this site is likely to be the result of an unusual conformation or sequence in the fourth hypervariable region of the V H domain. Therefore, the absence of similar cleavage in other myeloma proteins tested could be explained by differences in the sequence of their fourth hypervariable regions.

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb′2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb′s was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb′2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb′s; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb′2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.

IgM was recovered from the serum of a patient (Den) with Waldenstrom's macroglobulinemia by preparative electrophoresis and gel filtration. In the latter step three species were resolved, with sedimentation coefficients of 7.7S, 11.5S, and 19.0S. Molecular weight determinations showed that the three species were monomeric, dimeric, and pentameric IgM, respectively. The 19S and 11S species were not dissociated in 6M urea. Serologic, immunologic, and biochemical studies indicated that the constituent light and heavy chains of each species were the same. Light chains prepared from 19S IgM (Den) contained a second component identified as J chain. Light chains from 11S and 7S IgM (Den) did not contain this component.

The M-component in the serum of a patient with multiple myeloma and hyperviscosity syndrome was found to be an immunoglobulin of the IgG-1(K) class which crystallized spontaneously from the serum. After purification, the protein crystallized readily under a variety of conditions. X-ray diffraction studies indicated a rhombohedral lattice with cell dimensions: a R = 170 ± 5 Å, α E = 109 ± 2°. Assuming a molecular weight of 1.5 × 10 5 , the crystals have been calculated to contain 66 ± 3% solvent, and six molecules per unit cell.Screening of the protein for antibody-like activity against a wide range of antigens gave positive results in several instances, in particular when tested against gum Karaya. This reaction was inhibited by D-glucuronic acid.

Deletions in both the heavy and light chains of an IgG1/κ immunoglobulin have been defined by means of amino acid sequence analysis, peptide mapping, and characterization of the peptides formed by treatment of the intact molecule and its Fc fragment with cyanogen bromide. The presence of a normal Fc fragment has been confirmed, and the deletions shown to consist of the NH2-terminal 102 residues of the H chain, and 68 residues within the variable region of the L-chain.

Crystals of the plasmin Fc fragment of human immunoglobulin G have been examined in the electron microscope at various stages during dissociation and recrystallization. Two characteristic shapes are recognized in the micrographs. One is a double, concentric annulus in which the outer annulus is seen to consist of 12 masses disposed radially and attached to the inner annulus by short stalks. The centres of maximum density of the concentric annuli lie at radii of 40 Å and 75 Å respectively. The other shape seen frequently in micrographs is a rod-shaped object appximately 150 Å in diameter and varying in length from 200 to 1000 Å. The rods are composed of segments 70 Å, each divided longitudinally into 35 Å sections. It is believed that the segments represent side views of the annuli. A model of Fc consistent with these findings and with the present state of knowledge is presented.

Research Article| October 01 1971 A new enzymic fragment (Facb) of rabbit immunoglobulin G G E Connell; G E Connell 1Medical Research Council Immunochemistry Unit, Department of Biochemi8try, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Search for other works by this author on: This Site PubMed Google Scholar R R Porter R R Porter 1Medical Research Council Immunochemistry Unit, Department of Biochemi8try, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1971) 124 (5): 53P. https://doi.org/10.1042/bj1240053Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation G E Connell, R R Porter; A new enzymic fragment (Facb) of rabbit immunoglobulin G. Biochem J 1 October 1971; 124 (5): 53P. doi: https://doi.org/10.1042/bj1240053Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1971 London: The Biochemical Society1971 Article PDF first page preview Close Modal You do not currently have access to this content.

The partial sequence of the light chain of the myeloma-like immunoglobulin Sac shows a large deletion in its variable region. The sequence provides evidence that the corresponding gene was formed by the repair of DNA broken at nonhomologous positions. Data from other immunoglobulin (heavy) chains containing large deletions are compatible with their genes also being the result of DNA breakage and nonhomologous repair. Single homologous reciprocal exchanges in DNA networks at immunoglobulin loci could be the cause of the nonhomologous breaks. The relevance of these events to the generation of normal antibody variability remains to be determined.

The widely occurring enzyme γ-glutamyl cyclotransferase acts on γ-glutamyl peptides to effect the release of the terminal glutamyl residue as the cyclic derivative pyrrolidone carboxylic acid. The enzyme has been purified from pig liver by (1) ammonium sulfate precipitation from the supernatants of homogenates, (2) CM-cellulose treatment, (3) DEAE-Sephadex chromatography, and (4) preparative polyacrylamide gel electrophoresis. The homogeneity of the highly purified enzyme was demonstrated by ultracentrifugation and electrophoretic analyses. By means of (5) isoelectric focusing, two forms of the enzyme with isoelectric points 4.87 and 4.95 were separated. These forms proved to have very similar sedimentation velocities, molecular weights, and amino acid compositions, and to have the same amino acid, glycine, as the N-terminal residue.

An immunoglobulin IgG (Sackfield) which is known to have polypeptide chains shorter than those of typical proteins of its class has been subjected to fragmentation by papain in the presence of cysteine. One fragment was recovered which was indistinguishable from normal Fc fragment. The other fragment was related to normal Fab fragment but differed from it in several of its properties. The molecular weight was only one-half that of normal Fab. The optical rotatory dispersion spectrum of IgG (Sackfield) had features which differed from those of typical IgG myeloma proteins. The optical rotatory dispersion spectrum of Fc (Sackfield) was identical with those of other myeloma proteins, while the Fab (Sackfield) spectrum reflected the differences observed in the parent protein.

A protein of the Ig G family has been isolated from the serum of a patient with a tentative diagnosis of a plasma cell neoplasm. The protein has a lower sedimentation constant (5.4) and a lower molecular weight (125,000) than normal immunoglobulins of the G family. The protein has heavy-chain determinants of type G and light-chain determinants of the κ-type. Heavy and light chains have been prepared by reductive cleavage followed by gel filtration. The heavy-chain preparation is homogeneous in starch gels in acidic buffer containing urea but has a faster mobility than normal Ig G heavy chains. The light-chain preparation is resolved into two components in electrophoresis, and both have slower mobility than normal Ig G light chains. The heavy- and light-chain preparations cross react with normal Ig G heavy and light chains in immunodiffusion analysis. Sedimentation equilibrium studies suggest that both the heavy and light chains have lower molecular weights than their normal counterparts.