Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin.

We have synthesized two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin. Labeled oligonucleotides were used as hybridization probes to screen bacterial clones containing cDNA sequences primed with oligo(dT) and inserted into the plasmid vector pBR322. One beta 2-microglobulin cDNA clone was detected in the 535 bacterial plasmid clones that were screened. The clone has been characterized by blotting and nucleotide sequence analysis. The cloned beta 2-microglobulin sequence contains 217 base pairs of the 3' untranslated region of the mRNA and 328 base pairs (97%) of the coding region.     

Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen beta chain.

The HLA-D locus in the major histocompatibility complex controls the expression of the genetically polymorphic HLA-DR antigens. mRNA coding for the beta chains of these antigens was partially purified from the human lymphoblastoid cell line Raji. The mRNA was copied into double-stranded cDNA and cloned in Escherichia coli. One clone, pDR-beta-1, obtained by hybrid selection, carries a 1070-base-pair insert comprising all of the coding region except the signal sequence and a substantial portion of the untranslated region. To identify pDR-beta-1, highly purified HLA-DR antigen beta chains derived from Raji cells were subjected to NH2-terminal amino acid sequence determination. This sequence displayed extensive homology with that deduced from the nucleotide sequence at the 5' end of the pDR-beta-1 coding region. Taken together, the amino acid and nucleotide sequences strongly argue in favor of Raji cells containing at least two beta-chain loci.     

Three-dimensional structure of beta 2-microglobulin.

The three-dimensional structure of beta 2-microglobulin, the light chain of the major histocompatibility complex class I antigens, has been determined by x-ray crystallography. An electron density map of the bovine protein was calculated at a nominal resolution of 2.9 A by using the methods of multiple isomorphous replacement and electron density modification refinement. The molecule is approximately 45 X 25 X 20 A in size. Almost half of the amino acid residues participate in two large beta structures, one of four strands and the other of three, linked by a central disulfide bond. The molecule thus strongly resembles Ig constant domains in polypeptide chain folding and overall tertiary structure. Amino acid residues that are the same in the sequences of beta 2-microglobulin and Ig constant domains are predominantly in the interior of the molecule, whereas residues conserved among beta 2-microglobulins from different species are both in the interior and on the molecular surface. In the crystals studied, the molecule is clearly monomeric, consistent with the observation that beta 2-microglobulin, unlike Ig constant domains, apparently does not form dimers in vivo but associates with the heavy chains of major histocompatibility complex antigens. Our results demonstrate that, at the level of detailed three-dimensional structure, the light chain of the major histocompatibility class I antigens belongs to a superfamily of structures related to the Ig constant domains.     

Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.

The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families.     

Middle molecules removal. Beyond beta2-microglobulin

The uremic toxin removal capacity mainly depends on dialyzer and hemodialysis modes. The low-flux hemodialysis only removes solutes having molecular weights less than 5.000 Da. High-flux hemodyalisis represents a form of low-volume hemodiafiltration because of the internal filtration and back-filtration that can take place within a dialyzer. Hemodiafiltration with large volumes of replacement fluid seems to be the best technique for removing all small, medium-sized and large molecules. The objective of our study was to evaluate the large molecules removal bigger than beta2-microglobuline on high flux haemodialysis and on-line hemodiafiltration with postdilutional infusion, in patients with three times a week dialysis and on short daily dialysis. We studied 24 patients, 15 males and 9 females stable on haemodialysis programme, twelve on standard four to five hours three times a week dialysis and twelve on 2 to 2 1/2 hours six times a week dialysis. All patients were dialysed with Fresenius 4008 monitor, three sessions on high flux haemodialysis (HD) and three sessions on on-line hemodiafiltration (OL-HDF). Two sessions with each filter were performed (polisulfone HF80, polyethersulfone Arylane H9 and new polisulfone APS 900). Pre and postdialysis concentrations of urea, creatinine, (beta2-microglobulin (beta2-m), myoglobin, prolactin and alpha1 microglobulin (alpha1-m) were measured. There was no difference in urea and creatinine small molecules removal. beta2m removal was 68% on HD and 81% on OL-HDF. Myoglobin and prolactin present a similar removal pattern, a higher removal with new filters (60% with Arylane and 59% with APS) in comparison with clasical polisulfone (22% with HF80). The mean alpha1-m reduction rate on HD was 6% and on OL-HDF 22%. OL-HDF with APS 900 filter was the most remove technique (35.4%), significatively higher than the other modes and filters. We can conclude that the new filters generation reach a better uremic toxins removal, specially in large molecules higher than beta2-m and on HD modality.     

Crystallographic studies of bovine beta2-microglobulin.

Crystals of the bovine milk protein lactollin yield x-ray diffraction data extending to a resolution of 2.8 A. Lactollin is a bovine analogue of beta2-microglobulin, a protein that is homologous in amino acid sequence to the constant domains of immunoglobulins and is the light chain of the human and murine major histocompatability antigens. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 77.4, b = 47.9, and c = 34.3 A. The unit cell parameters and physical chemical solution studies indicate that the molecule exists in the crystal and in solution as a single polypeptide chain of 12,000 daltons.     

Cell-free synthesis and segregation of beta 2-microglobulin.

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.     

Relationship of beta2-microglobulin to arterial stiffness in Japanese subjects.

Beta2-Microglobulin (beta2m) is related to inflammatory diseases, but there have been few reports of a relationship between beta2m and atherosclerosis. We have examined the influence of beta2m on brachial-ankle pulse wave velocity (baPWV) to clarify whether it is related to arterial stiffness. baPWV, beta2m, C-reactive protein (CRP), and conventional risk factors were measured in 614 males and 158 females. The adjusted means of baPWV were compared with the quartiles of beta2m, and significant differences in baPWV were observed across the quartiles of beta2m (p = 0.037). After being adjusted for potential confounders, quartile 4 of beta2m, quartile 4 of CRP, and the combination of high beta2m plus high CRP were significantly associated with a high value of PWV (quartile 4 of beta2m: odds ratio [OR] 2.53, 95% confidence interval [CI], 1.31-4.89; quartile 4 of CRP: OR 2.27, 95% CI, 1.18-4.34; high beta2m plus high CRP: OR 5.60, 95% CI, 2.38-13.2). These results suggest that beta2m is associated with an increase of arterial stiffness. Further studies are needed to clarify whether beta2m is related to atherosclerotic diseases, and whether the combination of beta2m and CRP measurement is a useful predictor for the development of atherosclerosis.     

Two allelic forms of mouse beta 2-microglobulin.

Two allelic forms of mouse beta 2-microglobulin (beta 2m), the small polypeptide chain of H-2 histocompatibility antigens, have been identified. The two forms can be distinguished by NaDodSO4/polyacrylamide gel electrophoresis. All inbred mouse strains express a single beta 2m isotype. Mice heterozygous for beta 2m synthesize both forms, showing codominant expression of beta 2m alleles. In mice heterozygous for both H-2 and beta 3m, individual H-2 histocompatibility antigens associate with both beta 2m forms. Preliminary structural studies indicate differences in peptide composition between the two forms.     

Assignment of the gene for beta 2-microglobulin (B2m) to mouse chromosome 2.

We have assigned the gene (B2m) coding for murine beta 2-microglobulin (B2M) to mouse chromosome 2 by using a novel panel of Chinese hamster-mouse somatic cell hybrid clones. Because of 35 independent primary hybrids used in this study were derived from two types of feral mice, each with a different combination of Robertsonian translocation chromosomes, as well as from mice with a normal complement of acrocentric chromosomes, analysis of 16 selected mouse enzyme markers provided data on the segregation of all 20 mouse chromosomes in these hybrids. Mouse B2M was identified in cell hybrids by immunoprecipitation with a species-specific anti-mouse B2M antiserum followed by two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitated polypeptides. Enzyme analysis of the segregant clones excluded all chromosomes for B2m assignment except mouse chromosome 2, and karyotype analysis of nine informative hybrid clones confirmed the assignment of B2m to this chromosome. These results demonstrate that, in the mouse, as in man, B2m is not linked to the major histocompatibility or immunoglobulin loci.     

Isolation of cDNA clones for human complement component C2.

Two cDNA clones for complement component C2 have been isolated from a high-complexity human liver cDNA library by using a mixture of 64 synthetic oligonucleotides as a probe. The 400-base-pair insert of pC201 codes for a region containing the active site serine residue and the secondary substrate binding pocket of the serine protease. This part of C2 is 34% homologous to the corresponding region of the related serine protease factor B and additional similarity is evident from a number of conservative amino acid replacements in this region. The insert of pC201 was used as a specific probe in RNA transfer analysis to determine the size of the C2 mRNA as approximately equal to 2.9 kilobases. Southern blot analysis of genomic DNA of unrelated individuals identified a single C2 locus and showed no cross-hybridization with the factor B locus.     

Musculoskeletal manifestations in beta 2-microglobulin amyloidosis. Case discussion.

The cases presented illustrate some of the typical (case 1) and less common (case 2) clinical features of beta 2m amyloidosis. The accumulation of beta 2m amyloid in tissues is a potentially severe complication of dialysis-treated chronic renal failure. Beta 2m amyloidosis has been shown to have distinct clinical, radiologic, and pathologic features. The pathogenesis of this condition is not yet clearly understood, and recommendations for the clinical management of these patients at present are limited to recognition of the disease and symptomatic treatment. Further insights into the biology of this disease should lead to new strategies for prevention and treatment.     

Extension of A beta2M amyloid fibrils with recombinant human beta2-microglobulin.

In order to elucidate the pathogenesis of A beta2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of A beta2M amyloid fibril (fA beta2M) extension with native beta2microglobulin (n-beta2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant beta2M (r-beta2M) in vitro. n-Beta2M and r-beta2M were incubated with fA beta2M purified from synovial tissues excised from A beta2M amyloidosis patients. The fA beta2M extension reaction could be explained by a first-order kinetic model in both beta2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both beta2Ms. The fA beta2M extended with both beta2Ms assumed the similar helical filament structure, although the fibrils extended with r-beta2M were slightly wider than those extended with n-beta2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodified fA beta2M, we next extended fA beta2M repeatedly by the algorithmic protocol with r-beta2M. As the generation of the extended fibrils proceeded, the initial rate of the extension reaction increased The ultrastructure of fibrils was completely preserved throughout the repeated extension steps. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fA beta2M extended repeatedly with r-beta2M were composed solely of r-beta2M. The use of these r-beta2M and fA beta2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation of fA beta2M in vitro.     

Plasma levels of beta 2-microglobulin in rheumatoid arthritis.

A simple and inexpensive method is described for the determination of beta 2-microglobulin (beta 2-MG) by enzyme-amplified single radial immunodiffusion. The values obtained with this method correlate well with those determined by means of a commerical RIA kit. Using the immunodiffusion method we have measured the plasma levels of beta 2-MG in 135 patients with rheumatoid arthritis (RA) and normal serum creatinine levels. 33% of the patients had increased concentrations of beta 2 MG, but the levels were found to correlate poorly with the values of several variables generally used as indices of the degree inflammatory activity in RA. Furthermore, in contrast to earlier claims to the contrary, beta 2-MG correlated positively with age. The value of beta 2-MG in plasma as an index of inflammatory activity in RA is questioned.     

Allelic forms of beta 2-microglobulin in the mouse.

Spleen cells from BALB/c and C57BL/6 mice were cultured separately or together, and the biosynthetically labeled supernates were examined by two-dimensional polyacrylamide gel electrophoresis. Although there were no major labeled proteins in the mixed group that were not present in the separate cultures, there was a major low-molecular-weight protein that differed in charge in the two strains. This protein was identified as beta 2-microglobulin; it could be labeled with 125I on the cell surface by using the lactoperoxidase technique, was noncovalently attached to the H-2K molecule, and had the expected size and charge when compared with human beta 2-microglobulin. Both acidic and basic forms were present in (BALB/c X C57BL/6) F1 hybrids, suggesting codominant expression, although allelic exclusion was not ruled out. Either parental form could combine with one parental form of the H-2K molecule. The beta 2-microglobulin gene does not appear to be closely linked to either the H-2 or th immunoglobulin heavy-chain complexes. It is proposed that beta 2-microglobulin is an "effector subunit" of histocompatibility antigens and that its physiological role is to interact with a specific killing structure on the surface of cytolytic T lymphocytes and thereby initiate cell destruction.     

Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin.

Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing soluble protein, protofilaments and some fibrils or only fibrils in a solid phase system (ELISA) was modest with a slow kinetics compared to the positive delta IgG control. Soluble A beta induced no detectable CA in a liquid phase system (complement consumption assay) while fibrillar A beta caused CA at 200 mg/ml and higher concentrations. Soluble beta 2-microglobulin (beta 2M) purified from peritoneal dialysates was found to be as potent a complement activator as A beta in both solid and liquid phase systems while beta 2M purified from urine exhibited lower activity, a difference which may be explained by differences observed in SDS-resistant oligomers and isoforms. Soluble Amyloid A-protein caused no significant CA. A beta and beta 2M activated complement via the classical pathway. The modifying influence by amyloid-associated molecules on A beta-induced CA was also investigated, but neither serum amyloid P component nor heparan sulfate did significantly alter the A beta-induced CA. The results indicate that not only fibrillar A beta but also oligomers of, in particular, beta 2M from patients with dialysis-associated amyloidosis are capable of inducing CA at supra-physiological concentrations.     

Complete amino acid sequence of murine beta 2-microglobulin: structural evidence for strain-related polymorphism.

Primary structural analyses of beta 3-microglobulin isolated from the tumor cell lines EL4.BU (derived from a C57BL/6 mouse) and C14 (derived from a BALB/c mouse) have revealed the presence of an amino acid difference at position 85 of this molecule. beta 2-Microglobulin isolated from histocompatibility antigens of EL4.BU has alanine at this position, whereas that from C14 has aspartic acid. Determination of the sequence of these molecules has employed radiochemical methodology that was developed in studies of murine histocompatibility antigens. The sequence obtained in this study is: Ile - Gln - Lys - Thr - Pro - Gln - Ile - Gln - Val - Tyr - Ser - Arg - His - Pro - Pro - Glu - Asn - Gly - Lys - Pro - Asn - Ile - Leu - Asn - Cys - Tyr - Val - Thr - Gln - Phe - His - Pro - Pro - His - Ile - Glu - Ile - Gln - Met - Leu - Lys - Asn - Gly - Lys - Lys - Ile Pro - Lys - Val - Glu - Met - Ser - Asp - Met - Ser - Phe - Ser - Lys - Asp - Trp - Ser - Phe - Tyr - Ile - Leu - Ala - His - Thr - Glu - Phe - Thr - Pro - Thr - Glu - Thr - Asp - Thr - Tyr - Ala - Cys - Arg - Val - Lys - His - Ala/Asp - Ser - Met - Ala - Glu - Pro - Lys - Thr - Val - Tyr - Trp - Asp - Arg - Asp - Met. Comparison of the sequence of murine beta 2-microglobulin to the sequences reported for the homologues from man, rabbit, and guinea pig indicate identities of 68%, 66%, and 61%, respectively.