Simulation of pH-dependent edge strand rearrangement in human beta-2 microglobulin

Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.     

Genome-wide association study identified the human leukocyte antigen region as a novel locus for plasma beta-2 microglobulin

Beta-2 microglobulin (B2M) is a component of the major histocompatibility complex (MHC) class I molecule and has been studied as a biomarker of kidney function, cardiovascular diseases and mortality. Little is known about the genes influencing its levels directly or through glomerular filtration rate (GFR). We conducted a genome-wide association study of plasma B2M levels in 6738 European Americans from the Atherosclerosis Risk in Communities study to identify novel loci for B2M and assessed its association with known estimated GFR (eGFR) loci. We identified 2 genome-wide significant loci. One was in the human leukocyte antigen (HLA) region on chromosome 6 (lowest p value = 1.8 × 10^?23 for rs9264638). At this locus, 6 index SNPs accounted for 3.2 % of log(B2M) variance, and their association with B2M could largely be explained by imputed classical alleles of the MHC class I genes: HLA-A, HLA-B, or HLA-C. The index SNPs at this locus were not associated with eGFR based on serum creatinine (eGFRcr). The other locus of B2M was on chromosome 12 (rs3184504 at SH2B3, beta = 0.02, p value = 3.1 × 10^?8), which was previously implicated as an eGFR locus. In conclusion, although B2M is known to be a component of MHC class I molecule, the association between HLA class I alleles and plasma B2M levels in a community-based population is novel. The identification of the two novel loci for B2M extends our understanding of its metabolism and informs its use as a kidney filtration biomarker.     

An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.     

Tissue distribution of the lipocalin alpha-1 microglobulin in the developing human fetus.

Alpha-1 microglobulin (alpha(1)m), a lipocalin, is an evolutionarily conserved immunomodulatory plasma protein. In all species studied, alpha(1)m is synthesized by hepatocytes and catabolized in the renal proximal tubular cells. alpha(1)m deficiency has not been reported in any species, suggesting that its absence is lethal and indicating an important physiological role for this protein To clarify its functional role, tissue distribution studies are crucial. Such studies in humans have been restricted largely to adult fresh/frozen tissue. Formalin-fixed, paraffin-embedded multi-organ block tissue from aborted fetuses (gestational age range 7-22 weeks) was immunohistochemically examined for alpha(1)m reactivity. Moderate to strong reactivity was seen at all ages in hepatocytes, renal proximal tubule cells, and a subset of pancreatic islet cells. Muscle (cardiac, skeletal, or smooth), adrenal cortex, a scattered subset of intestinal mucosal cells, tips of small intestinal villi, and Leydig cells showed weaker and/or variable levels of reactivity. Connective tissue stained with variable location and intensity. The following cells/sites were consistently negative: thymus, spleen, hematopoietic cells, lung parenchyma, glomeruli, exocrine pancreas, epidermis, cartilage/bone, ovary, seminiferous tubules, epididymis, thyroid, and parathyroid. The results underscore the dominant role of liver and kidney in fetal alpha(1)m metabolism and provide a framework for understanding the functional role of this immunoregulatory protein.     

Predictive value of cystatin C and beta-2 microglobulin in preeclampsia

The purpose of this study was to determine whether the levels of cystatin C and beta-2 microglobulin (B2M) are altered during the second trimester in the plasma of women who subsequently develop preeclampsia.Study designWe performed a case control study to compare the levels of cystatin C and B2M in women in whom preeclampsia ultimately developed (n = 30) and in pregnant women who remained normotensive throughout gestation (n = 60). The maternal plasma levels of cystatin C and B2M were measured by enzyme-linked immunosorbent assay. Blood samples were collected between 15 and 20 weeks’ gestation for fetal aneuploidy screening and frozen at ?20 °C until assay after groups had been selected.ResultsThe median concentrations of cystatin C and B2M were significantly higher in those who subsequently developed preeclampsia when compared to those of normal pregnancy (median 668.6 ng/ml and 418.3 μg/ml vs 413.7 ng/ml and 321.2 μg/ml, respectively).ConclusionsIn this study, the maternal plasma levels of cystatin C and B2M were significantly elevated in pregnant women who subsequently developed preeclampsia as compared with normotensive women. Alterations of these proteins antedate clinical symptoms and, thus, they may be useful for early identification of patients at the risk of developing preeclampsia.     

Heparin binding induces conformational changes in Adeno-associated virus serotype 2

Adeno-associated virus serotype 2 (AAV2) uses heparan sulfate proteoglycan as a cell surface-attachment receptor. In this study the structures of AAV2 alone and complexed with heparin were determined to 18 Å resolution using cryo-electron microscopy and three-dimensional image reconstruction. A difference map showed positive density, modeled as heparin, close to the icosahedral twofold axes and between the protrusions that surround the threefold axes of the capsid. Regions of the model near the threefold place the receptor in close proximity to basic residues previously identified as part of the heparin binding site. The region of the model near the twofold axes identifies a second contact site, not previously characterized but which is also possibly configured by heparin binding. The difference map also revealed two significant conformational changes: (I) at the tops of the threefold protrusions, which have become flattened in the complex, and (II) at the fivefold axes where the top of the channel is widened possibly in response to movement of the HI loops in the capsid proteins. Ordered density in the interior of the capsid in the AAV2–heparin complex was interpreted as nucleic acid, consistent with the presence of non-viral DNA in the expressed capsids.     

Recognition of the alpha-1 and alpha-2 domains of H-2 molecules by allospecific cloned T cells

Previously we had shown that allospecific bulk cultures of cytolytic T lymphocytes lysed the products of cloned class I major histocompatibility genes expressed after DNA-mediated gene transfer. In these experiments, performed by using cloned allospecific T cell effectors, a T cell hybridoma, and recombinant DNA technology, we have been able to map determinants recognized by these T cell clones to the alpha-1 domain of H-2Dd and the alpha-2 domain of H-2Ld (four of eight clones). Target cells used were L cells (H-2k), expressing wild type or hybrid H-2 antigens of H-2d origin. Thus, for the first time determinants recognized by cloned T cells are found in the recombined alpha-1 and alpha-2 domains.     

Effects of β2–microglobulin mutations on the alpha-1 helical region of H2-Ld

 Beta-2 microglobulin (β₂m)has been shown to have an effect on the structural and functional constraints that facilitate proper class I antigen presentation. To date, no evidence has pinpointed the β₂m-specific amino acids that play an integral role in affecting structure in and around the peptide binding region of class I. To delineate β₂m amino acid positions that affect the alpha-1 helical region, we generated a series of mutant β₂m proteins bearing precise amino acid substitutions. The amino acid positions chosen were based upon previous results which demonstrated that human β₂m association with H2-L^d altered the structure of the alpha-1/alpha-2 super-domain. β₂m mutant proteins were used in β₂m exchange assays with cells expressing H2-L^d. Following exchange, cells were assayed to determine whether mutant β₂m association resulted in structural alteration of class I extracellular domains. The alteration in H2-L^d structure was evidenced by an increase in the binding of an antibody (34-1-2), specific for the alpha-1 helical region of H2-L^d. Results demonstrated that amino acid substitutions in β₂m positions 33 and 53 led to a dramatic increase in the reactivity of the alpha-1 domain-specific antibody 34-1-2. Identifying β₂m amino acid positions that influence the structure of the peptide binding region may allow for a better understanding of cellular immune responses that center upon class I/β₂m expression. Received: 18 December 1997 / Revised: 19 February 1998     

RNA induces conformational changes in the SF1/U2AF65 splicing factor complex

Spliceosomes assemble on pre-mRNA splice sites through a series of dynamic ribonucleoprotein complexes, yet the nature of the conformational changes remains unclear. Splicing factor 1 (SF1) and U2 auxiliary factor (U2AF⁶⁵) cooperatively recognize the 3′ splice site during the initial stages of pre-mRNA splicing. Here, we used small-angle X-ray scattering to compare the molecular dimensions and ab initio shape restorations of SF1 and U2AF⁶⁵ splicing factors, as well as the SF1/U2AF⁶⁵ complex in the absence and presence of AdML (adenovirus major late) splice site RNAs. The molecular dimensions of the SF1/U2AF⁶⁵/RNA complex substantially contracted by 15 Å in the maximum dimension, relative to the SF1/U2AF⁶⁵ complex in the absence of RNA ligand. In contrast, no detectable changes were observed for the isolated SF1 and U2AF⁶⁵ splicing factors or their individual complexes with RNA, although slight differences in the shapes of their molecular envelopes were apparent. We propose that the conformational changes that are induced by assembly of the SF1/U2AF⁶⁵/RNA complex serve to position the pre-mRNA splice site optimally for subsequent stages of splicing.     

The sodium gradient induces conformational changes in the renal phosphate carrier

Phosphate transport across brush border membranes from kidney cortex is very sensitive to inhibition by phenylglyoxal, an arginine modifier. Sodium-dependent phosphate influx into brush border membrane vesicles was inhibited by 60%. In contrast, phenylglyoxal had no effect on passive influx or on sodium-dependent efflux of phosphate. Preincubation of the vesicles with sodium prior to the addition of phenylglyoxal demonstrated a strong protective effect of intravesicular sodium (73% protection). Phosphate also protected the transporter from inhibition, but from the extravesicular side only (63%). Substitution of phosphate by sulfate offered no protection at all, indicating the specificity of protection. Addition of both substrates (sodium and phosphate) offered an additional protection from the extravesicular side compared to that offered by phosphate alone (92 versus 55%). There was no additional protection when both substrates were added to the intravesicular side. Phosphate influx measured in the presence of sodium but in the absence of a sodium gradient was totally unaffected by phenylglyoxal modification. There was no inhibition on phosphate influx measured in equilibrium exchange conditions. We propose a model for the phosphate carrier in which the sodium gradient induces a conformational change and an arginine residue is essential for the coupled flux of sodium and phosphate.     

Structural abnormalities in chromosome 15 in cell lines with reduced expression of beta-2 microglobulin

Using somatic cell hybrids, the gene for beta-2 microglobulin has been assigned to human chromosome 15. We found it of interest to study a number of human lymphoid cell lines in light of this finding, to analyze whether spontaneously occurring loss or reduction of beta-2 microglobulin could be correlated with any aberration in chromosome 15. The Daudi cell line was shown to be devoid of any beta-2 microglobulin in total extracts. Chromosome analysis showed that one of the two chromosomes 15 was deleted in the region q14q21 on the long arm; in some metaphases, both chromosomes were deleted in this region. The K562 cell line was found to express very low (if any) membrane-associated beta-2 microglobulin. Chromosome analysis showed that this line was near-triploid, with two normal chromosomes 15 and one translocation chromosome t(15;18) involving the long arm of chromosome 15, whereby the segment proximal to the breakage point in band q15 was lost. The Namalwa cell line showed a reduction in membrane-associated and total beta-2 microglobulin. Chromosome analysis showed this line to contain one chromosome 15 which was shorter than its normal homolog. The deletion could be identified as such in the region q14q21 in Daudi cells, but is probably somewhat smaller than the one in Daudi cells. Since analyses of beta-2 microglobulin production and chromosomes 15 on several other human cells failed to reveal any abnormalities in either of these respects, we postulate that genes responsible for beta-2 microglobulin synthesis and membrane expression could be located in the region q14q21 on the long arm of chromosome 15. Since beta-2 microglobulin associated with the membrane was found to be absent in the K562 line, where total beta-2 microglobulin was nearly as high as in cell lines with normal membrane expression, it is suggested that membrane expression of beta-2 microglobulin can be regulated independentlyAbbreviations used in this paper are BSS Earle's balanced salt solution - Q banding bands obtained by fluorescence with Quinacrine Mustard - R banding bands obtained by fluorescence with Acridine Orange - FITC fluorescein isothiocyanate - EBV Epstein-Barr virus     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

Endocytosis and dissociation of class I MHC molecules labeled with fluorescent beta-2 microglobulin

Membrane class I MHC molecules of Con-A activated and lymphoma murine cells have been labeled by exchange of the cell's beta 2m with soluble fl-beta 2m. It has previously been shown that this method of labeling is specific and does not affect the biologic properties of class I MHC Ag. With this labeling it has been possible to demonstrate the constitutive endocytosis of class I MHC by fluorescence microscopy and by measuring the resistance to quenching by crystal violet of the internalized fl-beta 2m molecules. We could also follow the kinetics of beta 2m dissociation from the class I molecules at different pH. At pH 5.5, that is the average pH of endosomes, there is considerable dissociation within 15 to 20 min, that is the average recycling half time of class I MHC containing endosomes in activated T cells. Inasmuch as the process is reversible it is likely that, in the recycling endosomes of T cells, class I MHC molecules undergo conformational changes with beta 2m going off and on and with consequent changes of the peptide binding site. This process might be involved in Ag presentation, but, because it is apparently limited to T cells, it would play a role in the presentation of the cell's own TCR in idiotypic interactions between T cells.     

Altered structure of HLA class I heavy chains associated with mouse beta-2 microglobulin

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin ( ₂m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse ₂-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human ₂m restores these reactivities. Since most of the amino acid differences between mouse and human ₂m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.