The amino acid sequence of human V-trace, a basic microprotein without known function, was determined by automated Edman degradations of the carboxynethylated polypeptide chain and of fragments obtained by cyanogen bromide treatment and tryptic digestion after blocking of lysine residues. The single polypeptide chain contained 120 residues, and the calculated Mr was 13,260. A proline residue at position 3 was partly hydroxylated. The presence of y-trace in a significant proportion of the cells in the anterior lobe of simian and human pituitary glands was demonstrated by immunohistochemical procedures with a rabbit antiserum against human V-trace. The tissue localization and amino acid sequence of V-trace indicated that this protein is connected with the peptidergic gastroenteropancreatic neuroendocrine system. Human y-trace is an alkaline, low Mr protein first described in 1961 as a constituent ofnormal cerebrospinal fluid and of urine from patients with renal failure (1-3). The biological role of ytrace is unknown, although the protein has been shown to occur in low concentration not only in cerebrospinal fluid and urine but also in plasma, saliva, and semen (4-6). A sensitive enzyme immunoassay for measurement of y-trace has been described (7). It allowed quantitation of the protein in all human fluids investigated and demonstrated that, in healthy adults, the concentration of y-trace in cerebrospinal fluid is 5.5 times that in plasma. Recent immunohistochemical studies of y-trace have shown it to be present in some human brain cortical neurons, in adrenal medulla, and in the A cells of the pancreatic islets (8-10). Some amino acid sequence similarities also have been found between human glucagon and the amino-terminal part of y-trace (8). Therefore, the question whether y-trace is related to the peptidergic gastroenteropancreatic neuroendocrine system has arisen. This report describes the complete amino acid sequence of y-trace and presents immunohistochemical evidence of the presence of the protein in a large number of cells in the pituitary gland. Experimental details of the work will be published separately.MATERIALS AND METHODS Isolation of V-Trace. Urine containing about 20 mg of y-trace per liter from patients with renal failure was collected in bottles containing the protease inhibitor benzamidinium chloride and the antimicrobial agent sodium azide. y-Trace was isolated from the pooled urine as described (8). Its purity was checked by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (11) and agarose gel electrophoresis (12).Structural Determination. Isolated y-trace was reduced and carboxymethylated with '4C-labeled reagent in 6 M guanidinium chloride. The carboxymethylated protein was then fragmented by cyanogen bromide treatment (13) or by tryptic digestion after reversible blocking ofamino groups by citraconylation (14). One ofthe cyanogen bromide fragments (no. 3) was further digested with Staphylococcus aureus V8 protease. On each occasion the fragments were separated by gel chro...
Gamma-Trace was purified in large amounts from urine and used for the production of a specific rabbit antiserum. An enzyme immunoassay for quantitation of gamma-trace was developed using the pure protein as a primary standard. Its sensitivity was approximately 30 microgram/l. An enzyme amplified single radial immunodiffusion was developed as well. Its sensitivity was approximately 0.3 mg/l. These assays allowed quantitation of gamma-trace in normal human biological fluids. The following results were obtained (mean +/- SD): cerebrospinal fluid: 5.8 +/- 2.2 mg/l, plasma: 1.1 +/- 0.42 mg/l, saliva: 1.8 +/- 0.88 mg/l and urine: 0.095 +/- 0.057 mg/l. Plasma samples from patients with advanced renal failure revealed gamma-trace values up to 13 times the normal mean plasma value. The results indicate a production of gamma-trace in the central nervous system and that the protein is primarily catabolized by the kidney.
Hereditary CNS amyloid angiopathy occurring in Icelanders is the first human disorder known to be caused by deposition of cystatin C amyloid fibrils in the walls of the brain arteries leading to single or or multiple strokes with fatal outcome. One or more affected members have been verified by histological examination in 8 families containing 127 affected. These originated from the same geographic area. Abnormally low value of cystatin C found in the cerebrospinal fluid of those affected can be used to support or make diagnosis of this disease, also in asymptomatic relatives. By amino acid sequence analysis the amyloid fibrils in the patients are found to be a variant of cystatin C (gamma-trace), a major cysteine proteinase inhibitor. The variant protein has an amino acid substitution (glutamine for leucine) at position 58 in the amyloid molecule. It is postulated that a point mutation has occurred leading to production of amyloidogenic protein causing the disorder.
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