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Partial characterization of the sialic acid-free forms of alpha-1-acid glycoprotein from human plasma.

ABSTRACT: Some of the properties of sialic acid-free alpha(1)-acid glycoprotein prepared by mild acid hydrolysis (pH1.6 at 80 degrees for 1hr.) were compared with those of neuraminidasetreated alpha(1)-acid glycoprotein. Chemically, the former contained less fucose (15%) and amide (2%) residues. Physicochemically, it had undergone certain changes primarily pertaining to the secondary structure, so that the specific optical rotation was more negative than that of the latter. A further expression of this change is probably the difference in the pH range of the resolution into two bands on electrophoresis. The resolution of the glycoprotein prepared by mild acid hydrolysis seems to be extended to more acidic pH values both by starch-gel and free moving-boundary electrophoresis. On ultracentrifugation both preparations appeared homogeneous and sedimented with a rate of 3s. Removal of sialyl residues at different pH values, in the range 1-7, showed that 2moles of sialic acid/mole of protein are very strongly bound. The two variants of alpha(1)-acid glycoprotein were isolated from pooled sialic acid-free alpha(1)-acid glycoprotein by preparative starch-gel electrophoresis and from selected blood of normal adults by fractionation by solubility and chromatography. Ultracentrifugal and starch-gel electrophoretic analyses at pH5, with incubation times of 1 or 24hr., demonstrated that no dissociation-association equilibrium (constant sedimentation coefficient and molecular weight) or isomerization (constant apparent electrophoretic mobilities) exist between the two variants. Therefore these variants are not sub-units of native alpha(1)-acid glycoprotein but represent modifications of naturally occurring proteins. Further, it was shown that the difference in the electrophoretic mobilities between the two variants was not due to any difference in amide content. Immunochemically, the two variants share the same determinants.

SUBMITTER: Schmid K

PROVIDER: S-EPMC1270595 | biostudies-other | 1967 Aug

REPOSITORIES: biostudies-other

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Project description:Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD.

Project description:Surface expression of alpha-(2,8)-linked polymers of sialic acid in adult tissues has been correlated with metastasis of several human cancers. One approach to chemotherapeutic intervention against the spread of these cancers involves the development of immunogenic molecules that elicit an antibody response against alpha-(2,8)-linked polysialic acids. Naturally occurring polysialic acids are not viable candidates because they are present during embryonic development and are recognized as self by the immune system. These natural polymers also have poor pharmacokinetic properties because they are readily degraded by neuraminidase enzymes. We have been interested in developing structural surrogates of polysialic acids in an effort to overcome these limitations. Reported herein are microwave-assisted solid-phase peptide syntheses and structural characterization studies of a series of alpha/delta hybrid peptides derived from Fmoc-Neu2en and Fmoc-Glu(OtBu)-OH. Conformational experiments including circular dichroism, NH/ND exchange, and ROESY in aqueous solution were performed to study the secondary structures of these hybrid foldamers. ROESY data were analyzed with the assistance of XPLOR-NIH that was modified to include parameter and topology files to accommodate unnatural amino acids and the delta amide linkages. The results indicate that stable secondary structure is dependent upon both the amino acid sequence and the configuration of Glu. The most stable foldamer was composed of a total of 6 residues beginning with L-Glu at the carboxy terminus and alternating Neu2en and L-Glu residues. In water, this foldamer adopts a right-handed helical conformation with 3.7 residues per turn, 7.4 A pitch, 5.8 A diameter, and a length of 18.5 A, which is stabilized by both classical C=O...H-N backbone interactions and by pyranose ring O and L-Glu HN H-bonding. These structural features orient the L-Glu carboxylates along the helical backbone with a periodicity that matches the carboxylate positions along the reported G2(+) left-handed helix of alpha-(2,8)-polysialic acid. However, the charge density of the foldamer is one-half that of the natural polymer. These findings provide a fundamental understanding of the factors that influence stable secondary structure in hybrid Neu2en/Glu systems, and the tools we have developed establish a viable platform for the rational design of alpha-(2,8)-polysialic acid surrogates.

Project description:Fibroblasts from patients with sialic acid storage disease (SASD), sialidosis, mucolipidosis II, and from normal controls, were incubated in the presence of the glycoprotein fetuin that was tritium-labelled in its sialic acid residues by the periodate/[3H]borohydride reduction method, and the fate of the intracellular radioactive sialic acid (C7-sialic acid) followed in pulse-chase experiments. The model glycoprotein was readily endocytosed and degraded, more than 90% of the radioactivity being trichloroacetic acid (TCA)-soluble after 4 days of incubation. In all of the patients' fibroblasts, there was an increased accumulation of TCA-soluble radioactivity and, upon chase, a much lower rate of elimination than in normal controls. Gel chromatography of the material from the chase experiment showed that, in normal cells, most of the radioactivity at zero time behaved as free C7-sialic acid. This, as well as material of larger size (sialyloligosaccharides), was very much diminished by 48 h. In cells from two patients with SASD, there were large peaks both in the sialic acid and oligosaccharide positions; whereas the oligosaccharides were somewhat decreased by the end of the chase period, the sialic acid was essentially unchanged. In sialidosis fibroblasts, the radioactive material consisted of oligosaccharides, but very little C7-sialic acid; the elimination of the oligosaccharides was retarded. In normal cells, about 80% of the radioactivity released into the medium after 48 h chase behaved as free C7-sialic acid upon gel chromatography and t.l.c. Subcellular fractionation in Percoll gradients showed that the radioactive C7-sialic acid remaining in normal cells after 48 h of chase was mainly localized in the cytosol. In SASD cells, on the other hand, it was associated with lysosomal fractions which, unexpectedly, exhibited an abnormally low density. Our findings demonstrate that SASD fibroblasts degrade the sialoglycoprotein but, unlike normal cells, accumulate the liberated C7-sialic acid along with sialyloligosaccharides in their lysosomes. The results therefore support the concept of a defective transport system for sialic acid in the lysosomal membrane of patients with SASD.

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Partial characterization of the sialic acid-free forms of alpha-1-acid glycoprotein from human plasma.

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