Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Species specificity in the isoelectric spectra of immunoglobulin light chains.

Light chains isolated from normal immunoglobulin give rise to a finite number of discrete bands when analyzed by isoelectric focusing in polyacrylamide gel. The positions and relative intensities of the bands were identical in light chains isolated from different individuals of a species, but clear differences were evident when light chains of several mammalian species were compared.     

Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.     

Messenger RNA for immunoglobulin light and heavy chains in MOPC-315 plasmacytoma and variants

Mouse plasmacytoma MOPC-315 produces light and heavy immunoglobulin chains. The variants, MOPC-315 NP-1 and MOPC-315 NR, synthesize only heavy or light chains, respectively. To eludicate the inability of MOPC-315 variants to produce intact light or heavy chains, complementary DNAs (cDNAs) to the mRNAs were prepared. From the kinetics of DNA-RNA hybridization, the RNA of the MOPC-315 NP-1 variant was shown to contain few or no sequences homologous with MOPC-315 light chain mRNA. Thus, the inability of this variant to produce light chain results from the absence of light chain mRNA. In contrast, the polyadenylated mRNA fraction of the MOPC-315 NR variant, which does not synthesize heavy chain, contains about two-thirds of the sequences present in heavy chain mRNA. Thus, this variant contains a fragment of heavy chain mRNA.     

Temporal relationship of translation and glycosylation of immunoglobulin heavy and light chains.

The initial glycosylation of MPC 11 gamma 2b heavy chains occurs quantitatively in vivo when the nascent heavy chains reach a size of approximately 38 000 daltons. Nonglycosylated, completed MPC 11 heavy chains cannot be glycosylated in these cells. Other classes of mouse heavy chains (i.e., mu, alpha, and gamma 1) also appear to be glycosylated as nascent chains; nonglycosylated, completed heavy chains cannot be glycosylated by the cell in any of these cases. In contrast, variant MPC 11 cells synthesizing a heavy chain with a carboxy-terminal deletion appear to glycosylate some heavy chains prior to chain completion and some heavy chains after chain completion and release from the polysomes. Similar to the variant MPC 11 cells, MOPC 46B cells (which synthesize a kappa light chain containing an oligosaccharide attached to an asparagine located 28 residues from the amino terminus) glycosylate the majority of light chains after prior to chain completion but also some light chains after chain completion and release from the polysomes. In addition, it appears that, although completed MOPC 46B light chains can be glycosylated if they are present in a monomeric form, they cannot be glycosylated if they are present in a covalent dimeric form.     

The identification of messenger RNA coding for immunoglobulin heavy and light chains of Xenopus laevis

Total poly(A)-containing mRNA isolated from Xenopus spleens was translated in a rabbit reticulocyte lysate in vitro protein-synthesizing system. Approx. 1% of the radioactivity incorporated into the protein was precipitated by an antibody directed against adult Xenopus IgM. The immunoprecipitated proteins were characterized as IgM heavy and light chains by their molecular weight as determined by polyacrylamide-sodium dodecyl sulfate gel electrophoresis The sequence variability of the synthesized light c hain proteins was analyzed by isoelectric focusing and shown to be indistinguishable from authentic Xenopus immunoglobulin light chain proteins derived from IgM. The data presented here identify Xenopus spleen mRNA as a potential source of a natural immunoglobulin mRNA population with which the development of the immune system can be studied.     

Comparative studies of two types of bovine immunoglobulin G heavy chains

;Fingerprints' of bovine colostrum and serum immunoglobulin G1 heavy chains were extremely similar, but different from serum immunoglobin G2 heavy chains. Serum immunoglobulin G1 and immunoglobulin G2 heavy chains were treated with cyanogen bromide. The fractions from the C-terminal end of the heavy chains were isolated and the amino acid sequence of this fraction from immunoglobulin G2 was:His-Glx-Ala-Leu-His-Asx-His-Tyr-Met-Gln-Lys-Ser-Thr-Ser-Lys-Ser-Ala-GlyThe amino acid composition of this fraction from immunoglobulin G1 was the same except for the methionine, which in immunoglobulin G1 was replaced by threonine.     

Disparity in the kinetics of onset of hypermutation in immunoglobulin heavy and light chains

The present paper describes a comparative analysis of light chains associated with primary and secondary IgM, as well as with secondary IgG antibodies to fluorescein, undertaken in order to explore the relationship between light chain somatic hypermutation and the isotype switch. The data reveal a disparity in the frequency of somatic hypermutation of secondary IgM heavy versus light chains. Among 20 secondary IgM light chains, a mutation frequency of 1/777 nucleotides was defined. In contrast, our previous analysis of the heavy chains of these molecules had identified a mutation frequency of 1/129. Among 17 IgG-derived light chains, obtained from animals killed at the same time point as those from which the secondary IgM antibodies were obtained, we measured a mutation frequency of 1/77. Finally, analysis of 20 light chains derived from primary IgM antibodies revealed a mutation frequency of only 1/1192 nucleotides. These data demonstrate that, prior to the class switch, light chain mutation occurs at a frequency considerably lower than that measured for the associated heavy chain gene. Six additional apparent mutations in the secondary IgM antibody 95B3 were all shared with a set of IgG antifluorescein antibodies belonging to the Vkappa 34 family. It is suggested that these light chains represent the products of a previously uncharacterized germ line gene.     

Addition of glucosamine and mannose to nascent immunoglobulin heavy chains.

We have investigated the process of protein glycosylation in an attempt to answer the question of whether glucosamine and mannose are added to nascent chains prior to chain completion or only to completed chains after release from the ribosome. The MPC 11 mouse plasmacytoma cell line used in these studies synthesizes a glycosylated gamma2b heavy chain which accounts for 12% of the total protein synthesis. Nascent chains were separated from completed chains by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. Our results indicate that both glucosamine and mannose are incorporated into nascent heavy chains prior to chain completion and release from the ribosome. Gel analysis of specifically immunoprecipitated nascent chains indicates that the carbohydrate moiety can be added to the nascent heavy chains very soon after the presumptive asparaginyl glycosylation site (CH2 domain) is synthesized on the ribosome.     

Primary structure of the variable regions of two canine immunoglobulin heavy chains.

The complete amino acid sequences of the variable regions of two canine immunoglobulin heavy chains have been determined by automated Edman degradation and found to be strongly homologous to the human VHIII subgroup. The canine sequences were identical with each other at 76 of 113 residue positions. Twenty-three of the 37 differences are located within the four hypervariable regions previously defined by the sequences of several human VHIII proteins. Forty-five of 77 framework residue positions are invariant in the seven human and two canine VHIII proteins which have been completely sequences. The canine proteins are 78% homologous to the framework of the human prototype. Phylogenetically associated residues before the first hypervariable region were confirmed and several potential phylogenetically associated residues were identified between the first and third hypervariable regions. This study represents the first complete amino acid sequences of VH regions of spontaneously occurring, nonhuman homogeneous immunoglobulins. The date demonstrate a high degree of preservation of VHIII structure in another species.