Purification of antibodies using protein L-binding framework structures in the light chain variable domain.

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L.

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.

Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain.

The gene for protein L, an immunoglobulin (Ig) light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, was cloned and sequenced. The gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-terminal region ("A") of 79 residues, the molecule contains five homologous "B" repeats of 72-76 amino acids each. Further, toward the COOH terminus, two additional repeats ("C") were found. These are not related to the "B" repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region ("W") was found, followed at the COOH-terminal end by a hydrophobic membrane anchor ("M"). Fragments of the gene were expressed, and the corresponding peptides were analyzed for Ig-binding activity. The B repeats were found to be responsible for the interaction with Ig light chains. An Escherichia coli high level expression system was adapted for the production of large amounts of two Ig-binding protein L fragments comprising one and four B repeats, respectively.

Rat alpha 1-microglobulin: co-expression in liver with the light chain of inter-alpha-trypsin inhibitor.

A 1162 bp rat liver cDNA clone encoding the immunoregulatory plasma protein alpha 1-microglobulin was isolated and sequenced. The open reading frame encoded a 349 amino acid polyprotein, including alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasma protein inter-alpha-trypsin inhibitor, 145 amino acids. The alpha 1-microglobulin/bikunin mRNA was found only in the liver when different tissues were examined. Free alpha 1-microglobulin and a polyprotein, containing both alpha 1-microglobulin and inter-alpha-trypsin inhibitor epitopes, were found in the microsomal fraction from rat liver homogenates.

Streptococcal protein G. Gene structure and protein binding properties.

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.

Maternal messenger RNA distribution in silkmoth eggs. I. Clone Ec4B is associated with the cortical cytoskeleton.

We have constructed a cDNA library from mature egg RNA of the silkmoth, Hyalophora cecropia. Differential screening of the library using cDNA made against mRNAs from the yolky cytoplasm (soluble fraction) and the cortical cytoplasm (cytoskeletal-associated or cortical fraction) resulted in several clones that hybridized to a higher degree to the cDNA from the cytoskeletal-associated fraction. We selected and analyzed the clone giving the strongest signal (designated Ec4b) for its distribution in situ and found that it bound to mRNAs in the nurse cell cytoplasm, in the cortex and in the follicle cells of oocytes. Hybridization of the insert from Ec4b to both detergent-soluble and -insoluble (cortical) RNA on dot blots further supported the observation that the mRNA corresponding to Ec4b was enriched in this cytoskeletal fraction. The mRNA for Ec4b was approximately 500 bases long and the gene seems to be a member of a large multigene family in the H. cecropia genome. Analyses of the nucleotide and amino acid sequences reveal similarity to lepidopteran chorion genes and a lesser but convincing similarity to vertebrate cytokeratins. The filter and in situ hybridization data point to the association of specific messenger RNAs with the cortical cytoskeleton of silkmoth oocytes. Aspects of the structure of the protein encoded by this mRNA suggest that it is a structural component necessary for formation of the cellular blastoderm of the embryo. The association of this maternal mRNA with the cortical cytoskeleton presents the interesting possibility that mRNA bound to the cytoskeleton may be capable of participating in the synthesis of new cytoskeleton or related structures during blastoderm formation.(ABSTRACT TRUNCATED AT 250 WORDS)

The genetics of insulin-dependent diabetes in the BB rat.

Very little is known about the genes involved in the pathogenesis of IDDM. One component is known to be linked to the major histocompatibility complex, but the other components are unknown. We know from the major animals models of IDDM, both the NOD mouse and the BB rat, that the disease is under multigenic control. However, due to the size and complexity of the mammalian genome as well as to the lack of useful clues, the location and identity of the other genes remains a mystery. This is compounded by the fact that well-characterized genetic markers are not available for all regions of the mammalian genome, and it is likely that at least some of the genes of interest are located in these regions. The testing of pedigrees for the linkage of RFLP with the genetic factors involved in IDDM promises to be the most effective means of mapping, and ultimately identifying, these genes. However, the number of genes which are theoretically necessary to test for linkage makes even this approach impractical. Here, we have described here how the amount of work and time can be significantly reduced by utilizing repetitive DNA sequences as probes for the linkage of random RFLPs to diabetes. With each screening, one can simultaneously test multiple unlinked loci in the genome. Preliminary results which show promising linkage to two of the genetic components have been presented, thereby supporting the usefulness of this approach.

The gene for the T lymphocyte alloantigen, RT6, is not linked to either diabetes or lymphopenia and is not defective in the BB rat.

In an outcross between a diabetic BB/H rat and a healthy Long Evans Hooded rat, the segregation of the RT6 gene was studied in the 207 F2 animals to look for linkage with diabetes or lymphopenia. The recessive gene, albino (c), was used as a marker for the RT6 gene because of the close proximity of these two genes on chromosome 1. Though most of the albino F2 rats should have been homozygous for the BB RT6 gene, we found no increase in the incidence of diabetes or lymphopenia among them when compared to their hooded littermates. Therefore, the RT6 gene was not linked to diabetes or lymphopenia in the BB rat. Moreover, the non-lymphopenic albino rats displayed normal RT6 expression when compared to the normal hooded rats showing that the RT6 gene from the BB/H grandfather was not defective. Any alteration in lymphocyte composition which could be specifically related to diabetes was studied by measuring all F2 rats for the major lymphocyte subsets including the RT6+ subset. We found that the typical pattern of lymphopenia described in diabetic BB rats was displayed by both diabetic and non-diabetic lymphopenic rats in the F2 generation. Thus, all these lymphocyte abnormalities including the depletion in RT6+ T lymphocytes appeared as a consequence of lymphopenia alone and could not be specifically related to diabetes.

Genetic studies in inbred BB/Wor rats. Analysis of progeny produced by crossing lymphopenic diabetes-prone rats with nonlymphopenic diabetic rats.

BB/Wor diabetes-prone (DP) rats are lymphopenic and frequently develop insulin-dependent diabetes. Diabetes-resistant (DR) BB/Wor rats are not lymphopenic and become diabetic rarely and at a significantly younger age. To examine the genetic basis for diabetes, lymphopenia, and age at onset of diabetes among inbred BB/Wor rats, we crossed nonlymphopenic diabetic rats with lymphopenic DP animals and studied F1, F2, and backcross progeny. F1 rats were neither diabetic nor lymphopenic. Diabetes (both types) and lymphopenia reappeared among F2 rats, confirming the permissive association of diabetes and lymphopenia and the recessive nature of both. The absence of diabetes in F1 rats also suggested that the combination of genes responsible for diabetes among lymphopenic and nonlymphopenic rats may be distinct. Nonlymphopenic parental, F1, and F2 rats revealed normal lymphocyte subsets, including CD8+ and RT6+ T-lymphocytes. Lymphopenic parental and F2 rats revealed the absence of CD8+ and RT6+ cells, indicating that these T-lymphocyte abnormalities of lymphopenic DP rats segregate with the lymphopenia gene. The distribution of the ages at onset of diabetes among F2 lymphopenic and F2 intercross rats was significantly earlier than among lymphopenic parental and backcross animals, suggesting that the age of diabetes onset is a heritable trait and that the gene(s) or genetic modifier(s) responsible for the earlier onset of F2 diabetes was acquired from the nonlymphopenic parents. Our genetic studies also confirmed the observations that the 2- and 7-kilobase Bam HI fragments of the MHC class I region do not correlate with diabetes or lymphopenia.

Protein G genes: structure and distribution of IgG-binding and albumin-binding domains.

Protein G (also designated Fc receptor type III) is the IgG-binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG-binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA-hybridization experiments to study the structure and distribution of the albumin- and IgG-binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG-binding region (I) and the other corresponded to repeats in the albumin-binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial species was found.

Derivation of non-lymphopenic BB rats with an intercross breeding.

Previous studies have suggested that the development of diabetes in the BB rats does not require the expression of T lymphopenia. In order to derive non-lymphopenic diabetic rats and define the relationship between the T cell abnormalities, MHC genotype, and diabetes, we performed a cross between BB/H and diabetes resistant BB/control followed by an intercross of the F1. In the F2, the overall incidence of diabetes and lymphopenia was 30% and 27%, respectively. Lymphopenia was strongly associated with diabetes (p less than 0.001) and was seen in 76% of the diabetic F2's. However, 6 of the diabetic were non-lymphopenic (24%) and 3 of the non-diabetics were lymphopenic (5%). In the non-lymphopenic diabetic animals, all T cell levels were within the normal range, but diabetes occurred at an earlier age than their lymphopenic littermates (p less than 0.001). In contrast to the strong association between the inheritance of lymphopenia and diabetes, no relationship between diabetes and Class I MHC restriction fragment length polymorphisms was found. We conclude: 1) Diabetes and lymphopenia are strongly associated inherited abnormalities in the BB rat and are not associated with Class I RFLP defined genotypes within the RTIu haplotype, 2) Animals in whom diabetes occurs in the absence of lymphopenia can be derived using this breeding approach 3) In our non-lymphopenic rats, diabetes occurred at an earlier age possibly reflecting the restoration of quantitative or qualitative T cell defects found in lymphopenic BB rats.

The molecular cloning of the chromogranin A-like precursor of beta-granin and pancreastatin from the endocrine pancreas.

The cDNA encoding the precursor form of the chromogranin A-related proteins, beta-granin and pancreastatin, was obtained by immune screening of rat insulinoma and pancreatic islet cDNA libraries. The sequence was virtually identical to that of rat adrenal chromogranin A, suggesting that the different molecular forms of chromogranin A immunoreactivity found in adrenal medulla and endocrine pancreas are related to differences in post-translational proteolytic processing. The rat chromogranin A, unlike its bovine and human counterparts, contained a 20-residue glutamine sequence inserted within the N-terminal beta-granin sequence. Although the encoding CA(G/A) repeat recurs frequently in the rat genome, the rat chromogranin A molecule appears to be the product of a single gene and mRNA transcript.

Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG.

Protein G is expressed at the cell surface of certain group C and group G streptococcal strains. The protein shows a unique and specific affinity for the Fc region of mammalian polyclonal and monoclonal immunoglobulin G (IgG). We have cloned the streptococcal gene coding for protein G into E. coli, using phage lambda as the vector. The protein G produced by E. coli infected with this phage was detected and analysed in Western blot experiments using radiolabelled IgG Fc fragments as a probe. Three major IgG Fc-binding bands were obtained corresponding to apparent mol. wts of 47,000, 57,000 and 65,000, respectively. Analysis of the expression in E. coli indicates that this heterogeneity is caused by a post-translational degradation of the molecule before lysis of the lambda infected E. coli cells occurred. The protein G produced in E. coli was purified by affinity chromatography on IgG-Sepharose followed by gel-filtration on Sephadex G-200. This highly purified E. coli-produced protein G was compared to protein G solubilized by papain from streptococci, in direct binding experiments and in a competitive binding assay. The two protein G variants were found to interact with polyclonal IgG from different species in a similar way. Streptococcal strains expressing protein G also show affinity for human albumin, and at the molecular level protein G was found to be responsible also for the binding of albumin. Thus, both E. coli-produced protein G and the proteolytic fragment of protein G obtained from streptococci, bound albumin. On the protein G molecule, two different and separate sites were found to bind IgG and albumin. Finally, when whole streptococci were incubated with human plasma, the interactions with protein G caused a coating of the bacteria with albumin and IgG, whereas other plasma proteins showed no affinity for protein G.

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans.

Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro) insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

Developmental and tissue-specific expression of alpha 1-microglobulin mRNA in the rat.

A rat liver cDNA library was constructed in the lambda gt11 expression vector. Three clones expressing alpha 1-microglobulin, an immunosuppressive plasma protein, were detected by screening with rabbit antiserum against rat alpha 1-microglobulin. The alpha 1-microglobulin activity from one of the clones, 6b, was confirmed with monoclonal antibodies in a solid phase radioimmunoassay. The nucleotide sequence of the fragment (165 base pairs) was determined, and the translated amino acid sequence (55 amino acids) showed a 75% homology to human alpha 1-microglobulin (position 122-176). Southern blots of restriction endonuclease-digested rat DNA indicated two distinct genes with alpha 1-microglobulin homology when probed with radioactive cDNA fragment from clone 6b. Northern blots showed the presence of a single mRNA species in rat liver, and the level was low in 1-month-old animals, increased to reach a maximum during adulthood (3 months), and decreased with aging (12 months). The alpha 1-microglobulin concentration in rat serum showed the same age dependence between 1 and 12 months, with the highest values at 3 months. Embryonic development (8.5-day to 17.5-day) was studied using total fetal RNA, and expression of alpha 1-microglobulin mRNA was detected in low amounts only at day 15.5. alpha 1-Microglobulin mRNA levels, studied by an RNA dot blot assay, were high in liver and kidney, low in brain and testis, and none were found in hypothalamus and spleen cells.

A deletion in a rat major histocompatibility complex class I gene is linked to the absence of beta 2-microglobulin-containing serum molecules.

Class I major histocompatibility antigens are composed of a heavy chain that is noncovalently associated with beta 2-microglobulin (beta 2m). Most class I molecules are membrane bound, but mouse and rat cDNA clones and genes without a functional code for the transmembrane amino acids have been identified. The membrane-associated class I molecules are important in the control of cell-mediated cytotoxicity, while the function of the soluble molecules remains unclear. Previous studies have shown that beta 2m circulates in rat serum in three different molecular weight classes. The first is free beta 2m (Mr, 12,000), the second is about Mr 70,000, and the third is roughly Mr 200,000. In an inbred subline of immunodeficient, diabetes-prone BioBreeding rats (BioBreeding/Hagedorn), previous work detected two restriction fragment polymorphisms in class I major histocompatibility complex genes, one of them a gene deletion on a 7-kilobase BamHI fragment and the other on a 2-kilobase BamHI fragment. In these rats we have found that the third serum beta 2m-binding size class is absent. Analysis of F1 and F2 individuals following cross-breeding between BioBreeding/Hagedorn rats and genetically related (nondiabetic) control BioBreeding w-subline rats demonstrated that the large-size serum peak of beta 2m was associated with the presence of the class I restriction fragments.