Human Urinary Proteins Research Articles

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein.

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.

Analysis of untreated human urinary proteins utilizing capillary zone electrophoresis

Analysis of untreated human urinary proteins utilizing capillary zone electrophoresis

Total Protein Determination in Urine: Elimination of a Differential Response between the Coomassie Blue and Pyrogallol Red Protein Dye-binding Assays

The total protein content of urine is a good index of renal function, but its determination is unreliable. Protein dye-binding assays are simple, but they characteristically lack a uniform response to different proteins. We investigated a differential response of the Sigma Microprotein Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to urine, using human albumin, albumin/globulin, or urinary protein as calibrator. The urine protein values (n = 60) obtained with the CBB assay were 110-13 500 mg/L (mean, 2390 mg/L) compared with 160-18 300 mg/L (mean, 3470 mg/L) obtained with the PRM assay (CBB:PRM protein concentration ratio, 0.46-0.88, mean, 0. 69 +/- 0.10). The differential response was highly reproducible as indicated by Sigma urine control Level 1 (within-day CBB:PRM ratio, 0.68 +/- 0.02; between-day CBB:PRM ratio, 0.67 +/- 0.04) and Sigma urine control Level 2 (within-day CBB:PRM ratio, 0.60 +/- 0.01; between-day CBB:PRM ratio, 0.59 +/- 0.02). The use of urinary protein as a calibrator (rather than human albumin) greatly improved the agreement between the assays when applied to urine (y(CBB) = 0. 972x(PRM) - 16 vs y(CBB) = 0.685x(PRM) + 17). In studies using urine controls, this calibrator also improved agreement between the CBB, PRM, trichloroacetic acid (TCA), and benzethonium chloride protein methods and, to a lesser extent, agreement with the TCA-Ponceau S method. The use of a urinary protein calibrator improves the agreement between different methods used to determine total protein in urine.

Proteoglycan core protein in human urine and its possible role on calcium oxalate urolithiasis.

There are only a few papers reporting on the role of proteoglycan core protein in calcium oxalate stone formation. The present study was carried out to investigate the role of core protein of proteoglycan in human urine on calcium oxalate (CaOx) crystallization. Proteoglycans were collected from whole human urine. The covalently bound glycosaminoglycans (GAG) of proteoglycans were then digested by GAG lyase. The inhibitory activity on CaOx crystal growth in vitro was measured before and after enzyme digestion of proteoglycans. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the core protein of proteoglycans and the analysis of amino acid sequence were performed. The core protein showed significant inhibitory activity on CaOx crystal growth, which scarcely changed when compared with that of proteoglycans before enzyme digestion. The SDS-PAGE revealed that the core protein was a single unit with a molecular weight of 26 kDa and amino acid sequencing demonstrated high homology to interalpha-trypsin inhibitor (ITI) light chain (bikunin) with Kunitz inhibitor domain as a core protein. The results suggested that human urine contains proteoglycans and a major part of them is ITI light chain (bikunin). The Kunitz inhibitor domain, a core protein of bikunin, has significant inhibitory activity on CaOx crystallization without GAG bound covalently to the core protein.

Quantitative Determination of Low and High Molecular Weight Proteins in Human Urine: Influence of Temperature and Storage Time

Many authors have studied the influence of storage on measured albumin concentrations in urine. Whereas some authors report that albumin concentrations decrease upon storage of urine at −20 °C (1)(2)(3)(4)(5)(6)(7)(8)(9)(10), others cannot confirm this (11)(12)(13)(14). As has been discussed extensively in the literature (15), these seemingly discrepant results might be explained by the use of pooled urine vs individual urine samples. The effect of storage may differ between various urine samples, probably because of variables such as protein concentrations, pH, and ionic strength. As a consequence, pooling of the urine samples might dilute the storage effect that can be found when using individual urine specimens. Other variables that have been tested for their influence on the measurements of urinary proteins are pH (1)(2)(3)(4)(13)(16)(17)(18), the tube material (4)(19)(20), handling of the sample (1)(7)(10)(12)(14), and the use of additives (21)(22)(23). It is likely that proteins other than albumin are also influenced by storage conditions, but information about the extent of such effects is limited (1)(3)(16)(17)(18)(21)(22)(23). In the study presented here, we measured several urinary proteins by nephelometry [albumin, transferrin, IgG, and α1-microglobulin (α1-m)] or by an RIA [β2-microglobulin (β2 …

Reference material "total protein in human urine"

Two batches of a reference material (RM) “total protein in human urine” were prepared for quality assurance in the determination of human urinary proteins. The material was prepared by freeze-drying of a pooled urine sample obtained from healthy volunteers. Protein concentrations in both batches were within physiological values, close to their upper limit. Interlaboratory comparisons conducted in 26 laboratories using the standard operation procedures elaborated for six of the most frequently employed methods (biuret, Bradford, Exton, Lowry, Pesce, and Watanabe) revealed that the results obtained by the individual methods were significantly different due to various reaction mechanisms involved. It was therefore necessary to certify the mean values and their uncertainties individually for each method. These operationally defined certified or information values are valid only when the prescribed standard operation procedures are strictly followed.

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak UP2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP2S(pkII) consisted of human urine proteins. Further analysis of UP2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP2S and UP2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP2S led to the generation of MoAbs which could bind both SEA and UP2S.

Clinical analysis of human urinary proteins using high resolution electrophoretic methods.

The application of isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.

Clinical application of capillary electrophoresis to unconcentrated human urine proteins.

Urine protein electrophoresis can be performed by capillary electrophoresis using spun unconcentrated urine diluted with running buffer. The separation which utilises the excellent sensitivity of capillary electrophoresis gives electropherograms similar to those obtained by diluting concentrated urine with buffer. A 72 cm x 50 microns internal diameter (ID) fused silica capillary was used for the analysis which took less than 15 min. Bence Jones protein can be detected from unconcentrated urine over urine protein concentrations ranging from 9.7 g/L to 0.04 g/L. Other clinical patterns, such as glomerular proteinuria, the presence of intact immunoglobulin and Tamm Horsfall protein can also be detected. The benefit of using unconcentrated urine is the time and cost saved by not concentrating the urine.

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP 2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP 2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak P 2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP 2S(pkII) consisted of human urine proteins. Further analysis of UP 2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP 2S and UP 2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP 2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP 2S led to the generation of MoAbs which could bind both SEA and UP 2S.

Spectroscopic characterization by photodiode array detection of human urinary and amniotic protein HC subpopulations fractionated by anion-exchange and size-exclusion high-performance liquid chromatography

A procedure for spectroscopic characterization and partial fractionation of human protein HC populations by high-performance liquid chromatography-photodiode array ultraviolet-visible detection is reported. Human protein HC from urine or amniotic fluid fractionated by anion-exchange HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as judged by the asymmetric elution pattern, consisting of a continuous irregular broad peak with several shoulders distributed along the whole chromatogram. Selected fractions containing shoulders were rechromatographed and finally six symmetrical homogeneous peaks with different retention times were obtained from each protein HC preparation. The direct automatic absorption spectra analyses at each peak maximum, indicated that all of the homogeneous peaks seemed to be protein HC, all of them associated to the same chromophore although with different stoichiometry ratios. Isoelectric focusing showed that each peak was composed of a limited number of subpopulations of protein HC with different isoelectric points. Size microheterogeneity has been also demonstrated in both urinary and amniotic protein HC preparations by a combination of size-exclusion HPLC on a TSK 3000 SW6 column and photodiode array detection. Partial fractionation of human albumin on an analytical anion-exchange Mono-Q PC 1.6/5 column, has allowed the identification of heterogeneous chromophore-containing populations displaying significant absorption in the visible region in resemblance to that of protein HC.

Two-dimensional electrophoresis of human urinary proteins following concentration by dye precipitation.

The use of dye precipitation for concentration of proteins prior to 2-DE has been demonstrated by application to human urine. The precipitation methods (T. Marshall and K. M. Williams, Clin. Chem. 1993, 39, 2314-2318; T. Marshall et al., Electrophoresis 1995, 16, 28-31) have been adapted for two-dimensional electrophoresis (2-DE) by modifying the reagent composition and adding sodium dodecyl sulphate (SDS) to the assay mixture. These modifications extend the precipitation range of the methods and overcome the problem of soluble glycoprotein-dye complexes. The protein-dye complex is recovered by centrifugation and dissolved in a small volume of sample denaturing buffer. The dye separates from the protein on 2-DE (forming a sharp band at the anodal end of the isoelectric focusing gel) so that the positional coordinates of the polypeptides are unaffected by the treatment. Dye precipitation provides a simple, rapid and highly economic method for concentrating urines of low to intermediate protein content (0.02-0.50 g/L) prior to 2-DE analysis.

A Novel Protein Assay Method Using Tetraphenylporphin tetrasulfonate (TPPS4)

Abstract A sensitive protein assay method which involves the reaction of TPPS4 with protein is described. When protein is added to TPPS4 solution, an absorption band with the maximum at 488 nm appears and the absorbance is proportional to the concentration of protein. Just Like the Soret absorption of the porphyrin, the new band is very narrow and there is no overlap at all between them, which means the free dyes would not give any background for the detection of the protein-TPPS4 complexes. A new spectrophotometric method for determination of protein has been constructed and applied to the determination of human plasma protein and urinary protein; The assay using microtiter plates has also been studied.

NH2-terminal sequence of novel pancreatic stone protein-related protein in human urine.

In a previous report, we detected the novel PSP-related protein in urine from a patient with diabetic nephropathy. This protein was different from PSP S1 and PSP S2-5 on the elution points of Mono S chromatography. To investigate the NH2-terminal sequence, we purified this protein by reverse phase chromatography and performed amino acid analysis. The results showed that the NH2-terminal sequence of this protein was one residue (arginine) longer than that of PSP S1.

Electrophoretic, chromatographic and immunological studies of human urinary proteins.

Urinary proteins from both sexes were analyzed by high resolution two-dimensional gel electrophoresis (2-DE). For well reproducible 2-DE patterns, the samples were concentrated and desalted in one step by vacuum dialysis. A reference map for urine proteins was established by the analysis of urine from 10 healthy persons. Proteins in urine that share immunogenicity with serum proteins were identified by use of antibody to whole human-serum protein in an affinity-column fractionation of urine and differential analysis of the adsorbed (serum component) and unadsorbed (non-serum component) fractions. For identification of individual proteins, coelectrophoresis, immunoblotting and affinity chromatography with corresponding antibodies were used. Proteins identified in the map, besides known serum proteins, included: the subunit of Tamm-Horsefall protein, the secretory component of IgA, constant breakdown products of alpha 1-antitrypsin and retinol-binding protein, the five isoforms of the beta chain of human chorionic gonadotropin and the subunit of prostatic acid phosphatase. In addition, we could demonstrate three proteins which are markedly pronounced in female urine, especially pregnant women. To get more information about the native properties of various urinary proteins, they were separated into four main peaks according to their sizes using fast protein liquid chromatography equipment. Possible interpolypeptide disulfide bonds were studied using a nonreducing 2-DE system. 2-DE in combination with other methods seems to be a valuable tool for the characterization of urinary proteins in defined renal or extra-renal diseases. An example is given by analyzing the immune complexes from seven patients with a urinary tract infection.

Identification and quantitation of human urine proteins by capillary electrophoresis

Capillary electrophoresis, as a fully automatable, powerful analytical technique, has the potential for use in clinical laboratories for separation and identification of protein types in normal and abnormal human urine specimens. Abnormal urine specimens containing previously identified proteins, anion-exchange resin treatment and addition of known components were used to identify and determine migration times of the peaks found in the electropherogram of human urine. The correlation coefficients between capillary electrophoresis and the currently used method of high-resolution agarose gel electrophoresis for Bence Jones protein and albumin were found to be 0.95 and 0.93, respectively.

Occurrence of two molecular forms of human acid sphingomyelinase.

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions.

Quantitation of Glutathione Transferase-Pi in the Urine by Radioimmunoassay

A radioimmunoassay procedure for the quantitation of glutathione transferase-pi was developed in order to determine the levels of this protein in human urine. The enzyme was isolated from human placenta with a purification factor of 366 (compared to the original high-speed supernatant fraction), and upon gel electrophoresis, only a single band was seen. Polyclonal antisera were subsequently raised in rabbits and found to be suitable for a radioimmunoassay. Glutathione transferase-pi was localized immunohistochemically to the cells of the distal tubules, the thin loop of Henle and the collecting ducts in the kidney. In contrast, the alpha-isoenzyme was localized exclusively in the proximal tubular epithelium. Samples of urine from healthy individuals contained about 6 ng of the pi-transferase/ml. The method proved to be specific for glutathione transferase-pi, and no cross-reaction with the alpha- or mu-transferase or with other proteins occasionally appearing in urine occurred. The protein was quite stable upon storage and insensitive to variations in the urine pH. Thus, it appears that glutathione transferase-pi can be conveniently quantitated by radioimmunoassay and changes in the concentration of this protein in human urine thus monitored.

Immunological characterization of pancreatic stone protein in human urine

In order to study the mechanism and origin of urine pancreatic stone protein (PSP), PSP was analyzed in the urine and sera from healthy subjects, patients with renal disease, and intensive care patients by Mono S chromatography and Western blotting. The elution patterns could be classified into three types. In control urine, a single peak of immunoreactive PSP (peak I) was identified at the position of PSP-S2-5 (type A). In three of seven patients with renal disease, another peak of urine immunoreactive PSP (peak II) was recognized at the position slower than that corresponding to that of PSP-S1 (type B). In urine from one patient with diabetic nephropathy, a third peak of immunoreactive PSP (peak III) was eluted between peaks I and II (type C). In Western blotting, the bands in urine from patients with renal disease and of those in ICU mainly appeared at the positions of high-molecular-weight types of PSP and PSP-S2-5, respectively. These results suggest that the kidney can be another major source of urine PSP in addition to the pancreas.

Macrophage-induced cytotoxicity and anti-metastatic activity of a 43-kDa human urinary protein against the Lewis tumor.

Resident, inflammatory or bone-marrow macrophages from C57Bl/6 mice incubated in vitro with a pure human urinary protein (HGP.43) decreased the growth rate of Lewis tumor cells (3LL). This inhibition of 3LL growth was the result of a cytotoxic activity of these macrophages which was independent of oxygen metabolites and nitrous oxide. Murine monoclonal antibodies (MAbs) against HGP.43 inhibited macrophage-mediated cytotoxicity. This cytotoxic activity was not due to the release of cytotoxic factors in the culture supernatant, showing that a contact between macrophages and tumor cells was required to express cytotoxicity. The presence of HGP.43 was absolutely necessary during the incubation of macrophages with target cells. In vivo, in HGP.43-treated mice, the growth of the primary tumor was not delayed but the size and number of lung metastases were significantly reduced 21 days after tumor inoculation.