Beta2-microglobulin causes abnormal phosphatidylserine exposure in human red blood cells

The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2-microglobulin accumulation in chronic uraemic patients.     

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.     

Beta(2)-microglobulin removal by extracorporeal renal replacement therapies

There is increasing evidence that end-stage renal disease patients with lower beta(2)-microglobulin plasma levels and patients on convective renal replacement therapy are at lower mortality risk. Therefore, an enhanced beta(2)-microglobulin removal by renal replacement procedures has to be regarded as a contribution to a more adequate dialysis therapy. In contrast to high-flux dialysis, low-flux hemodialysis is not qualified to eliminate substantial amounts of beta(2)-microglobulin. In hemodialysis using modern high-flux dialysis membranes, a beta(2)-microglobulin removal similar to that obtained in hemofiltration or hemodiafiltration can be achieved. Several of these high-flux membranes are protein-leaking, making them suitable only for hemodialysis due to a high albumin loss when used in more convective therapy procedures. On-line hemodiafiltration infusing large substitution fluid volumes represents the most efficient and innovative renal replacement therapy form. To maximize beta(2)-microglobulin removal, modifications of this procedure have been proposed. These modifications ensure safer operating conditions, such as mixed hemodiafiltration, or control albumin loss at maximum purification from beta(2)-microglobulin, such as mid-dilution hemodiafiltration, push/pull hemodiafiltration or programmed filtration. Whether these innovative hemodiafiltration options will become accepted in clinical routine use needs to be proven in future.     

The three-dimensional structure of beta2 microglobulin: results from X-ray crystallography

beta2-microglobulin, the light chain component of the major histocompatibility complex I, is involved in the development of DRA, an amyloid deposition disease occurring in man. Specifically, the beta2-microglobulin component, dissociated form the complex heavy chain, gives rise to amyloidogenic deposits in the joints of patients exposed to long dialysis periods. beta2-microglobulin three-dimensional structure is based on an antiparallel beta-barrel fold, with immunoglobulin domain topology, displaying structural flexibility in the crystal and NMR structures so fare determined. The structural bases of amyloidogenic potential in beta2-microglobulin can be related to local unfolding, to the tendency to aggregate laterally through non-compensated beta-strands, and partly also to its trend towards N-terminal proteolytic degradation. Such trends emerge quite clearly from inspection of a limited number of crystal structures of beta2-microglobulin as an isolated chain, separated form the major histocompatibility complex I heavy chain.     

Laboratory and sonographic findings in dialyzed patients with bilateral chronic knee pain versus dialyzed asymptomatic patients

The aim of this study is to evaluate connection of plasma level of beta2-microglobulin, C-reactive protein and uric acid as well as sonographic parameters like thickness of synovial membrane, thickness of femoral condylar cartilage and presence of joint effusion and Baker's cysts with bilateral knee pain in dialyzed patients, comparing them with parameters in asymptomatic dialyzed patients. Plasma levels of beta2-microglobulin and C-reactive protein were significantly higher in symptomatic patients while uric acid level showed no difference among the groups. In symptomatic patients synovial membrane was thicker and in those patients there were more knee effusions and Baker's cysts. Thickness of femoral condylar cartilage showed no difference between groups. That suggests that inflammatory mechanisms developing from beta2-microglobulin accumulation could be important factor in bilateral knee pain in dialyzed patients even in shorter duration dialysis.     

Cleaved beta 2-microglobulin partially attains a conformation that has amyloidogenic features.

beta(2)-Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of beta(2)-microglobulin (cleaved after Lys(58)) that as a trimmed form (Lys(58) is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that beta(2)-microglobulin cleaved after Lys(58) readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed beta(2)-microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why beta(2)-microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process.     

Isolation and immunoenzyme determination of human beta 2-microglobulin

Two procedures for isolation of homogeneous beta 2-microglobulin from urine of patients with systemic lupus erythematosus were developed: a procedure for isolation of beta 2-microglobulin with two-stage gel chromatography on Sephacryl S-200 and a procedure for isolation of homogeneous beta 2-microglobulin with affinity chromatography on rabbit polyclonal antibodies. The microglobulins isolated with the two procedures showed identical physicochemical properties and were used in development of a competitive immunoenzymatic assay method for determination of beta 2-microglobulin in the blood. The method was approved for determination of beta 2-microglobulin in blood serum of patients.     

血β2微球蛋白水平在系统性红斑狼疮中的临床意义

目的：探讨系统性红斑狼疮（systemic lupus erythematosus,SLE）患者血β2-MG水平与疾病活动的相关性及其临床意义。方法：随机抽取2012年2月-2012年7月我科收治的62例SLE患者（SLE组）和同期在我院门诊体检的健康体检者40例（对照组）,检测和比较两组血清β2-MG、自身抗体、补体水平,并对SLE患者进行SLEDAI评分,分析SLE患者血清β2-MG水平与自身抗体、补体水平和SLEDAI评分的相关性。结果：SLE组血β2-MG水平（3．11±0．40μg／mL）显著高于对照组（2．23±0．23μg/mL）,差异有统计学意义（P〈0．05）;其中发生口腔溃疡、浆膜炎及蛋白尿的SLE患者的血β2-MG水平与无此三种表现的患者相比显著升高,差异有统计学意义（P〈0．05）。SLE患者的血β2-MG水平与抗ds—DAN抗体、SLEDAI均呈显著正相关（分别为r=0．289,r=0．361,P〈0．01）,与C3呈负相关（r=-0．271,P〈0．05）。结论：SLE患者血β2-MG水平高于正常,可作为SLE疾病活动指标用于监测疾病活动。     

Beta2-microglobulin removal by extracorporeal renal replacement therapies

There is increasing evidence that end-stage renal disease patients with lower beta₂-microglobulin plasma levels and patients on convective renal replacement therapy are at lower mortality risk. Therefore, an enhanced beta₂-microglobulin removal by renal replacement procedures has to be regarded as a contribution to a more adequate dialysis therapy. In contrast to high-flux dialysis, low-flux hemodialysis is not qualified to eliminate substantial amounts of beta₂-microglobulin. In hemodialysis using modern high-flux dialysis membranes, a beta₂-microglobulin removal similar to that obtained in hemofiltration or hemodiafiltration can be achieved. Several of these high-flux membranes are protein-leaking, making them suitable only for hemodialysis due to a high albumin loss when used in more convective therapy procedures. On-line hemodiafiltration infusing large substitution fluid volumes represents the most efficient and innovative renal replacement therapy form. To maximize beta₂-microglobulin removal, modifications of this procedure have been proposed. These modifications ensure safer operating conditions, such as mixed hemodiafiltration, or control albumin loss at maximum purification from beta₂-microglobulin, such as mid-dilution hemodiafiltration, push/pull hemodiafiltration or programmed filtration. Whether these innovative hemodiafiltration options will become accepted in clinical routine use needs to be proven in future.     

beta2-Microglobulins: isolation, properties, and distribution.

beta2-Microglobulin is structurally related to immunoglobulin domains and is identical to the light chain of histocompatibility (HL-A) antigens. Similar to free light chains of immunoglobulins, beta2-microglobulin is most easily isolated from urine. We have previously purified human beta2-microglobulin from urine of patients with renal tubular resorption defects. Corresponding proteins have now been obtained from urine of rabbits and guinea pigs treated with sodium chromate. Sequence studies have established that the rabbit protein is rabbit beta2-microglobulin. The guinea pig protein closely resembles the human and rabbit beta2-microglobulins in amino acid composition, charge, molecular size, and also in the presence of an apparently analogous disulfide loop. These findings indicate that this protein is the guinea pig homologue of beta2-microglobulin. Physical-chemical studies suggest that human beta2-microglobulin and isolated immunoglobulin domains are similar not only in amino acid sequence but also in three-dimensional structure. Both types of molecules are compact and globular in shape and apparently contain beta-pleated sheet conformation. beta2-Microglobulin is present in free form in various body fluids and as a subunit of histocompatibility antigens on cell surfaces. Current estimates suggest that the number of beta2-microglobulin molecules on cell surfaces is higher than the number of histocompatibility (HL-A) antigens. Accordingly, beta2-microglobulin is possibly a subunit of additional cellular antigens or receptors.     

Purification and partial characterization of a new 85 KDa amyloidosis-related protein in chronic hemodialysis

An 85 KDa protein was purified by a multistep procedure (ultracentrifugation, HPLC, SDS-PAGE) from sera and amyloid deposits of patients on chronic hemodialysis and was characterized as a novel protein on the basis of its NH2 terminus (KVQLVE-V). This protein was formed by two subunits with Mr of 55 and 30 KDa and had affinity for Thyoflavin T, a fluorescent dye which was employed for labelling the protein prior HPLC. The 85 KDa was the only fluorescent component of ultracentrifugates from the serum of hemodialyzed patients while in amyloid fibrils it coexisted in roughly equimolar amounts with beta 2-microglobulin. This new high molecular weight protein which accumulates in uremia, could be co-responsible with beta 2-microglobulin for hemodialysis-related osteoarticular amyloidosis.     

Beta 2-microglobulinaemia: a sensitive index of diminishing renal function in diabetics.

A sensitive single measure of diminishing renal function is of importance in attempts to modify the progression of diabetic nephropathy. In 12 insulin-dependent diabetics with proteinuria plasma concentrations of beta 2-microglobulin were found to correlate more closely than plasma creatinine concentrations or creatinine clearance with glomerular function as measured by clearance of 52Cr-EDTA. The plasma beta 2-microglobulin concentration was raised in all patients with diminished glomerular filtration rate (below 80 ml/min/1.73 m2). By contrast, in two of these patients plasma creatinine concentration was normal. Plasma beta 2-microglobulin concentrations were stable throughout the day and not affected by food intake, unlike plasma creatinine concentrations, which rose in the afternoon and evening and after a meat meal. Plasma beta 2-microglobulin concentrations were the same in venous and capillary blood, the capillary blood being readily self-collected. Concentrations of beta 2-microglobulin were stable for up to 24 hours when whole blood was stored at 4 degrees C; adding aprotinin inhibited loss of beta 2-microglobulin for up to seven days. The results of this study suggest, therefore, that measuring beta 2-microglobulin concentrations is a simple and accurate method of detecting minor degrees of renal impairment and monitoring the effects of treatment.     

Evidence for linkage of murine beta 2-microglobulin to H-3 and Ly-4

Murine beta 2-microglobulin exists in 2 electrophoretically distinct forms; C57BL/6 mice possess the basic allele whereas BALB/c, CBA, AKR, and NZB possess the acidic allele. Mice heterozygous for beta 2-microglobulin express both alleles. Analysis of recombinant inbred mice suggests linkage of beta 2-microglobulin to H-2 or H-3. B10.C (28NX) mice (which possess the H-3c allele of BALB/c on a C57BL/10 background) possess the acid allele. Taken together, these results are consistent with the beta 2-microglobulin gene lying on chromosome 2, and being linked to H-3 and Ly-4.     

Clinical significance of urinary excretion of beta 2-microglobulin in patients with chronic glomerulonephritis]

Assuming, that deterioration of renal function in the primary glomerulonephritis may also result from the destructive lesions to parenchymal tissue of the renal cortex (with simultaneous lesions to renal tubuli), the assessment of the urinary excretion of beta 2-microglobulin could be approached as an indicator of renal function in this situation. The study involved 85 patients with chronic glomerulonephritis treated with immunosuppressors++. In these subjects beta 2-microglobulin concentration per one unit of the urine (UB2M) together with fractional excretion of this globulin (FEB2M) was studied. Statistically significant differences in mean UB2M and FEB2M values have been noted between patients with remission and with the lack of the remission following therapy. Gradual increase in UB2M values as well as FEB2M values have been noted with deterioration of excretory renal function. The results have shown, that the assays of UB2M, and particularly FEB2M, is a sensitive marker of proximal renal tubuli function and indirectly of the processes which take place in extraglomerular renal cortex structures. It might be there fore of prognostic value.     

Diurnal variations of serum beta 2-microglobulin in multiple myeloma

The chronobiological circadian behaviour in serum levels of beta 2-microglobulin has been investigated in three groups of subjects: (A) 6 healthy controls; (B) 6 patients with untreated multiple myeloma; (C) 6 patients with multiple myeloma in complete remission after polychemotherapy. From all subjects, under the same standard life conditions, venous blood samples were drawn at 4-hour intervals starting from midnight during the span of a whole day. Circulating serum beta 2-microglobulin levels were determined by RIA method. The time-related data were analyzed by chronograms and the "mean-group cosinor" method. A significant circadian rhythm for serum beta 2-microglobulin was detected in the control group, with a peak in the morning hours, and in untreated patients, with a peak in the afternoon hours. No significant rhythm was found in treated patients with multiple myeloma. A significant mesor reduction was noted in patients with complete remission, correlated with the absence of circadian rhythm, in respect to untreated patients. These data suggest that serum levels of beta 2-microglobulin could be related to the neoplastic plasma cell proliferation and to the effect of therapy, and that the circadian evaluation could be used as a guide in monitoring myeloma patients.     

A new form of amyloid protein associated with chronic hemodialysis was identified as beta 2-microglobulin

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.     

Studies on biosynthesis, assembly and expression of human major transplantation antigens

Biosynthesis and regulation of expression of transplantation as detected by a monoclonal antibody to HLA-A,B,C antigens (human leucocytic antigen) and a polyclonal antiserum to beta 2-microglobulin have been investigated using radioactive amino acids and sugars to label human lymphoid cells. We found unbalanced synthesis of HLA heavy chains and beta 2-microglobulin, the latter being in excess and secreted to the extracellular medium. In DAUDI cells, which are defective in beta 2-microglobulin, no HLA-A,B,C could be detected intracellularly even in the presence of added beta 2-microglobulin. Treatment of BRI-8 cells with tunicamycin, an antibiotic which inhibits glycosylation of polypeptides, almost had no effect on the levels of beta 2-microglobulin, while it markedly decreased that of HLA heavy chains, both on the cell surface and intracellularly. Glycosylation of the HLA heavy chains appeared to be an essential requirement for the normal expression of HLA-A,B,C antigens. The translation in vitro in a messenger-dependent reticulocyte system with total polysomes obtained from BRI-8 cells showed that beta 2-microglobulin was synthesized as a precursor. This larger polypeptide was converted into mature beta 2-microglobulin when protein synthesis was performed with microsomes instead of polysomes.     

Evaluation of selected indicators of the renal proximal tubule function in patients treated with gentamicin]

The study aimed at evaluating proximal renal tubule function in patients with nephrolithiasis and chronic pyelonephritis, and in patients with infectious diseases treated with gentamicin. The study involved 2 groups of patients: group A--17 patients with nephrolithiasis and chronic pyelonephritis and group B--30 patients with other infectious diseases (pneumonia, biliary tract infections) but with normal glomerular filtration rate. Patients from both groups were treated with gentamicin in a daily dose of 2-3 mg/kg for 7-10 days. Serum and urine creatinine levels were assayed in all patients prior to, 2-3, 7, 10 days, and after the treatment. Patients assigned to group B were divided into two subgroups: B1 included 15 patients with normal beta 2-microglobulinuria, and B2 15 patients with increased renal loss of beta 2-microglobulin and decreased tubular reabsorption of this protein. Significant increase in beta 2-microglobulinuria was seen on the third day of therapy, the decrease in the tubular reabsorption and glomerular filtration rate were noted in all patients on the seventh day of gentamicin administration. Beta 2-microglobulinuria was significantly higher in patients from groups A and B2 in comparison with group B1 in which no dysfunction of the proximal renal tubule was present before gentamicin therapy. A degree of beta 2-microglobulinuria is an early and sensitive indicator of gentamicin nephrotoxicity. The risk of nephrotixic symptoms is particularly obvious in patients with deteriorated function of renal proximal tubuli before the treatment with gentamicin.     

Serum beta 2-microglobulin in patients with monoclonal gammopathies

Beta-2-microglobulin concentrations were determined in serum samples from 45 patients with benign and malignant monoclonal gammopathies. In the group of patients suffering from multiple myeloma or Waldenstr?m macroglobulinemia the mean beta 2-microglobulin level was significantly higher than in the group with monoclonal gammopathy of undetermined significance. Values above 3 mg/L were highly indicative of a neoplastic process and were observed in all the Waldenstr?m patients and in greater than 90% of myeloma patients. No significant correlation was noticed between beta 2-microglobulin and monoclonal protein levels in any of the groups examined.     

The absence of β 2-microglobulin in Daudi cells: Active gene but inactive messenger RNA

The Daudi cell line is characterized by an absence of HLA antigen on its surface. This has been attributed to a lack of beta 2-microglobulin (beta 2m) while the heavy chain of HLA is present intracellularly. Karyotype analysis of Daudi cells has shown a deletion involving one of the beta 2-microglobulin alleles. It was generally believed that the absence of beta 2-microglobulin in that cell line resulted from an absence of expression of the remaining gene. We report here the unexpected finding of a normal amount of beta 2-microglobulin messenger RNA in Daudi cells. This was demonstrated by "Northern blot" hybridization with cDNA plasmid clones as a probe. This mRNA, however, when purified by hybridization-selection with beta 2-microglobulin plasmid DNA, is unable to function as messenger in protein synthesis and is therefore an inactive mRNA. The finding of a translationally inactive beta 2-microglobulin mRNA provides a new explanation for the absence of beta 2-microglobulin and therefore of HLA antigens in Daudi cells.