A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.     

Isolation and identification of hydroxyproline analogues of bradykinin in human urine

Hydroxyproline (Hyp) analogues of bradykinin and lysyl-bradykinin, in which the third residue of bradykinin, proline, is replaced by hydroxyproline, were isolated from human urine. Their amino acid sequences were confirmed by both amino acid and sequence analyses, and also by comparison of their chromatographic behavior with that of synthetic peptides. The possibility that Lys-Ala3-bradykinin, isolated by Mindroiu et al. [(1986) J. Biol. Chem. 261, 7407-7411] from human urine, was actually Lys-Hyp3-bradykinin is discussed.     

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.     

Isolation and characterization of novel phosphate-containing sialyloligosaccharides from normal human urine

Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc(alpha)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates.     

Isolation and characterization of N6-succinyladenosine from human urine.

From the urines of colon carcinoma patients and normal subjects we have isolated a nucleoside in which an amino group of aspartic acid is attached to the six position of purine ribonucleoside. The structure, N6-succinyladenosine, N-(9-B-D-ribofuranosylpurin-6-yl)aspartic acid was assigned on the basis of spectral data, chemical degradation, and by synthesis. The ultraviolet and mass spectra, chromatographic and electrophoretic mobilities, and the chemical properties of the naturally occurring nucleoside were identical to those of the synthetic N6-succinyladenosine. In contrast to the methylated and hypermodified nucleosides which are products of RNA catabolism, this urinary nucleoside appears to be derived from adenylosuccinic acid, a key intermediate required in the biosynthesis of ubiquitous, natural purine nucleotide adenosine-5'-monophosphate (AMP).     

Isolation and partial characterization of a low molecular weight trypsin inhibitor from human urine

A low molecular weight glycoprotein which completely inhibited trypsin at a 1 : 1 molar ratio was isolated from human urine. It was generated from a precursor molecule which in turn derived from plasma inter-alpha-trypsin inhibitor. It had one polypeptide chain with a molecular weight of about 20 000 and a high content of half-cystine residues. Its amino-terminal amino-acid sequence was Val-Thr-Glu-Val-Thr-X-Leu-Glu-Asp-.     

Isolation and characterization of human urine extracellular vesicles

Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO₄ prior to vesicle isolation by differential centrifugation. Treatment with ZnSO₄ did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.     

Isolation and characterization of calcium oxalate crystal growth inhibitors from human urine

Four acidic polypeptides which inhibit the growth of calcium oxalate crystals have been isolated from normal human urine, and two of these have been characterized with respect to their amino acid and carbohydrate compositions. SDS-Polyacrylamide gel electrophoresis of either of the two latter inhibitors revealed one prominent band that migrated with an apparent molecular weight of 17,500 daltons. γ-Carboxyglutamate is present in these inhibitors, and they contain a total of more than 25% glutamic and aspartic acids and less than 10% of basic amino acids.