Ulrich Wurster

Purification and Chemical Characterization of β-Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase

Abstract: β‐Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23–29 kDa was determined for the polypeptide on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Amino‐terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA24. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of β‐trace protein as prostaglandin D synthase [prostaglandin‐H2 D‐isomerase; (5Z, 13E)‐(15S)‐9α, 11 a‐epidioxy‐15‐hydroxyprosta‐5,13‐dienoate D‐isomerase; EC 5.3.99.2]. A conservative amino acid exchange (The instead of Ser) was detected at amino acid position 154 of the β‐trace polypeptide chain in the corresponding tryptic peptide. The two N‐glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29and Asn56 bear exclusively complex‐type oligosaccharide structures (partially sialylated with α2–3‐ and/or α2–6‐linked N‐acetylneuraminic acid) that are almost quantitatively α1‐6 fucosylated at the proximal N‐acetylglucosamine; ∼70% of these molecules contain a bisecting N‐acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.

Carbohydrate Structures of β-Trace Protein from Human Cerebrospinal Fluid: Evidence for “Brain-Type”N-Glycosylation

Abstract: The carbohydrate structures of β‐trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N‐linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N‐glycans were studied by compositional and methylation analyses, high‐pH anion‐exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N‐acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N‐acetylglucosamine residues of nearly all structures were found to be α1,6‐fucosylated. Peripheral fucose (about 20%α1,3‐linked to N‐acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N‐acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N‐acetylglucosamine only were present. At least 20 different N‐glycans were identified. Analysis of the site‐specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N‐acetylglucosamine, as well as terminal N‐acetylglucosamine and galactose residues, and significant amounts of N‐acetylneuraminic acid in α2,3 linkage), this protein can be classified as “brain‐type” glycosylated.

Differentiation of proteinurias with electrophoresis

In 1940, Ltitscher [1] and, independently, Longworth and MacInnes [2] described the use of electrophoresis for the analysis of proteinurias in patients with renal diseases. Almost 25 years later, Davies [3] and Ornstein [4] published the theory and practice of disc electrophoresis allowing the differentiation of urinary proteins according to their molecular weight. In the following years, many variations [5-23] of the polyacrylamide gel electrophoresis (PAGE) principle have been developed for use in nephrology (Table 1) and numerous publications have classified proteinurias according to different urinary protein molecular weight patterns [22, 24-31] . However, problems remain and many paediatric and adult nephrologists do not use PAGE routinely. In this issue of the journal, Brocklebank et al. [23] report the use of a commercially available, automated electrophoretic system (Phast system, Pharmacia, Uppsala, Sweden) to distinguish glomerular, tubular and a mixed gtomerulotubular pattern of proteinuria in children with various renal diseases. The method was used to separate urinary proteins in 1 gl unconcentrated urine taken from healthy or diseased children. Patients with a steroid-responsive nephrotic syndrome were found to excrete mainly albumin, transferrin and an albumin dimer, whereas some children with a steroid-resistant nephrotic syndrome in addition excreted two large molecular weight proteins, namely haptoglobin and IgG. Low molecular weight proteinuria was found in children with proximal renal tubular disorder. In the past, analysis of urinary proteins by electrophoretic methods was confined to well-equipped laboratories with experience of gel casting and processing. The introduction of the Phast system has now increased access to sodium dodecyl sulphate (SDS)-PAGE. As Brocklebank et al. [23] point out, the technique is easy to use in clinical practice and results are available in 2 h. However, the short separation distance of only 4 cm does not allow resolution of closely adjacent bands. Since the application volume is

ENZYME ACTIVITIES AND PROTEIN CONCENTRATION IN THE INTRAOCULAR FLUIDS OF TEN MAMMALS

An attempt was made to establish normal values for the total protein concentrations and the enzyme activities of LDH, MDH and PGI in the intraocular fluids of rats, guinea pigs, rabbits, cats, dogs, sheep, cattle, pigs, horses and humans. Remarkably little species differences were noted in 9 of the 10 mammals with vitreal enzyme activities falling into a narrow range between 8.4 U/l (PGI, horse) and 92.4 U/l (MDH, guinea pig). All species obeyed the sequence aqueous < vitreous < serum with exception of the rat, where vitreous activities surpassed serum at least two‐fold. The very low enzyme activities in the aqueous and vitreous humours demand special precautions against contaminations which can either derive from post‐mortem storage or inadequate sampling procedures. Conversely elevated enzyme activities could be used as probes for pathological processes or as a check for a clean preparation.

Bedeutung und Herkunft der Enzyme im Glaskörper des Rindes

The presence of malatedehydrogenase, alkal. phosphatase, GOT- and GPT-aminotransferases and of all glycolytic enzymes in the vitreous body of cattle was demonstrated. As the enzyme patterns of vitreous body and its cells show remarkable similarity, the serum pattern differing significantly, the cortex cells presumably represent the origin of the enzymatically active soluble proteins in the vitreous. Enzyme distribution in vitreous shows the same features as in other connective tissues thus indicating its mesenchymal nature. The “phospho-triose-glycerate-group” of enzymes has been found in the same constant proportions as in other tissues. The activities of its extracellular enzymes could be sufficient to account for the minimal metabolic requirements of the vitreous. Im Glaskörper des Rindes wurden sämtliche Enzyme der Glykolyse, Malatdehydrogenase, die Transaminasen GOT und GPT sowie alkal. Phosphatase nachgewiesen. Die Enzymmuster des Glaskörpers und seiner Zellen entsprechen einander, während die Verteilung im Serum deutlich abweicht, weshalb die Hyalocyten als hauptsächlicher Ursprungsort für die enzymatisch aktiven löslichen Proteine des Glaskörpers angenommen werden. Die Ähnlichkeit des Enzymmusters mit anderen Bindegeweben unterstreicht die mesenchymale Natur des Glaskörpers. Wie alle bisher untersuchten Gewebearten weist auch Glaskörper konstante Proportionen der Enzyme der „Phospho-Triose-Glycerat-Gruppe“ auf. Die Aktivität der extracellulären Enzyme könnte ausreichen, um den minimalen Stoffwechsel des Glaskörpers zu bewerkstelligen.

[Isoenzymes of lactic dehydrogenase in the vitreous body of cattle. Comparison with other tissues of the eye and serum (author's transl)].

The activities of LDH, MDH, GOT, PGI and CHE have been determined in the vitreous body and adjacent tissues of cattle-eye. LDH-Isoenzymes were separated with agar gel electrophoresis. Isoenzyme 1 of vitreous accounts for 65.4%, 2 for 14.3, 3 for 7.5, 4 for 6.4, 5 for 6.3%. Though this clearly demonstrates a heart-type pattern, an oxidative metabolism should not be concluded. Vitreous body rather belongs to the metabolically restricted erythrocytes, platelets and lens-fibers which depend on anaerobic glycolysis despite an aerobic LDH pattern. Since there is good agreement between the (iso-)enzyme ratios of vitreous body and hyalocytes, the cortex cells are thought to represent the origin of vitreous enzymes. Im Glaskörper und den angrenzenden Geweben des Rinderauges wurden die Enzymaktivitäten von LDH, MDH, GOT, PGI und CHE gemessen. Auftrennung der LDH-Isoenzyme des Glaskörpers mit der Agargelelektrophorese ergab für Bande 1: 65,4%, 2: 14,3, 3: 7,5, 4: 6,4 und Bande 5: 6,3%. Obwohl Glaskörper damit ein Herz-Typ-Muster aufweist, sind Rückschlüsse auf eine oxidative Stoffwechsellage nicht berechtigt. Vielmehr ist Glaskörper den in ihren metabolischen Fähigkeiten stark reduzierten Erythrocyten, Thrombocyten und Linsenfasern zuzurechnen, die mit einer H-Typ LDH glykolysieren. Aufgrund der guten Übereinstimmung zwischen Glaskörper und Hyalocyten im (Iso-)Enzymmuster werden die Glaskörperrindenzellen als Herkunftsort der Enzyme im Glaskörper postuliert.

ANTI-MOG ANTIBODIES

ABSTRACT There is a pressing need for biomarkers for diagnosis, prognosis and the potential efficiency of the diverse therapeutic options of the human autoimmune disease multiple sclerosis (MS). Antibodies to myelin oligodendrocyte glycoprotein (MOG) have been implicated to be involved in the pathogenesis of MS on the following considerations: (i) localization of MOG on the external surface of myelin, (ii) presence of abs to MOG peptides and protein in demyelinating plaques, and (iii) striking similarity in the disease course and pathological picture between MS and the MOG-induced animal disease experimental allergic encephalitis. Most research on MOG abs has been carried out with denatured antigen preparations representing the extracellular part of MOG by ELISA and Western Blot (WB). Such assays will detect primarily abs to linear epitopes, which are rarely pathogenic and may have arisen as a result of myelin breakdown. Depending on the assay conditions, the isotype IgG or IgM, and the definition of the cut-off frequencies from 38 to 100% have been reported. In contrast, positive results with fluid phase methods like FACS or RIA and native antigen stayed below 10%. The high rates of WB MOG positives among healthy controls prevent its use for diagnostic purposes, but a recent report suggested excellent performance as a prognostic test. However, up to now four follow-up studies have been unable to confirm the original optimistic results. The inherent problems of the WB MOG assay raise serious doubts if it will ever be applicable for routine patient testing.

Two-Month Follow-Up of the Changes in Vitreal Constituents after Argon Laser Coagulation of the Retina in Rabbits

The response of the vitreous after panretinal argon laser photocoagulation was followed over a period of 60 days. Two phases can be discerned. An an immediate reaction to the disturbance of the blood-retinal barrier at the site of the pigment epithelium the total protein content and malate dehydrogenase activity rise sharply within 3 days, 4- to 5-fold above a normal. The more protracted increase of lysosomal enzyme activities which reach their peak heigh after 14 days only, can be attributed to the action and proliferation of phagocytic cells. 2 months after treatment all parameters have returned to their normal levels.