Non-neuronopathic Gaucher disease due to saposin C deficiency

Gaucher disease is generally caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The degradation of glycosphingolipids requires also the participation of sphingolipid activator proteins. The prosaposin PSAP gene codes for a single protein which undergoes post-translational cleavage to yield four proteins named saposins A, B, C and D. Saposin (SAP-) C is required for glucosylceramide degradation, and its deficiency results in a variant form of Gaucher disease. In this report, we present clinical, biochemical, and molecular findings in a 36-year-old man and his 30-year-old sister with non-neuronopathic Gaucher disease due to SAP-C deficiency. Very high levels of chitotriosidase activity, chemokine CCL18, and increased concentration of glucosylceramide in plasma and normal beta-glucosidase activity in skin fibroblasts were observed in the patients. A molecular genetics study of the PSAP gene enabled the identification of one missense mutation, p.L349P, located in the SAP-C domain and another mutation, p.M1L, located in the initiation codon of the prosaposin precursor protein. The presented findings describe the first cases where the non-neuronopathic Gaucher disease has been definitely demonstrated to be a consequence of SAP-C deficiency. Three previously described cases in the literature displayed a Gaucher type 3 phenotype.     

鞘脂激活蛋白原的研究进展

鞘脂激活蛋白原（prosaposin）是一种多功能的糖蛋白。Prosaposin刚合成时其分子量为53kD，经过翻译后加工成为两种功能完全不同的蛋白：一种分子量为65kD，是4种鞘脂激活蛋白（sphingolipid activator protein，saposin）的前提物，在sortilin蛋白的作用下运送至溶酶体，由蛋白水解酶水解成为4种低分子量的鞘脂激活蛋白saposin A、B、C、D，其功能是作为鞘脂糖水解途径中所涉及到的溶酶体酶的激活剂，参与带有短的寡糖链的脂类的代谢；另一种分子量为70kD，prosaposin作为完整的糖基化的分泌蛋白出现，存在于一些生物体液中，例如精液、乳汁、脑脊液，并具备与鞘脂激活蛋白完全不同的功能。     

Trichinella spiralis secretes a homologue of prosaposin

Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin, the precursor of sphingolipid activator proteins (saposins) A-D originally defined in vertebrates. The protein contains four saposin domains, with the six cysteine residues which form the three intramolecular disulphide bonds in close register in each case. It differs substantially from vertebrate prosaposins in the N-terminal prodomain, the region separating saposins A and B, and completely lacks the C-terminal domain which has been demonstrated to be essential for lysosomal targetting in these organisms. The protein is secreted in unprocessed form with an estimated mass of 56 kDa, and contains a single N-linked glycan which is bound by the monoclonal antibody NIM-M1, characteristic of the TSL-1 antigens which are capped by tyvelose (3,6-dideoxy-D-arabinohexose). Immuno-electron microscopy localised the protein to membrane-bound vesicles and more complex multi-lamellar organelles in diverse tissues including the hypodermis, intestine and stichosomes, although it was absent from the dense-core secretory granules typical of the latter. Possible functions of a secreted prosaposin are discussed.     

Mutational analysis in a patient with a variant form of Gaucher disease caused by SAP-2 deficiency

It is now clear that the lysosomal hydrolysis of sphingolipids requires both lysosomal enzymes and so-called sphingolipid activator proteins (SAPs). One gene, called prosaposin, codes for a precursor protein that is proteolytically cut into four putative SAPs. These four SAPs, of about 80 amino acids, share some structural features but differ somewhat in their specificity. Domain 3 of prosaposin mRNA contains the coding region for SAP-2, an activator of glucocerebrosidase. While most patients with Gaucher disease store glucosylceramide due to defects in glucocerebrosidase, a few patients store this lipid in the presence of normal enzyme levels. In this paper we describe the identification of a point mutation in domain 3 of a patient who died with this variant form of Gaucher disease. Polymerase chain reaction amplification was performed in the small amount of genomic DNA available using primers generated from the intronic sequence s surrounding domain 3. The patient was found to have a T-to-G substitution at position 1144 (counting from the A of ATG initiation codon) in half of the M13 recombinant clones. This changes the codon for cysteine₃₈₂ to glycine. His father and unaffected brother also had this mutation, but his mother did not. She was found to have half of the normal amount of mRNA for prosaposin in her cultured skin fibroblasts. Therefore, this child inherited a point mutation in domain 3 from his father and a deficiency of all four SAPs coded for by prosaposin from his mother.     

Heat Shock Proteins in Hypoxic-Ischemic Brain Injury: A Perspective

There is much to suggest that the induction of heat shock protein synthesis is an important response to injury and stress in the brain. The role of heat shock proteins in neurological disease has been approached from two points-of-view. First, the induction and synthesis of specific proteins after brain cell injury provide a window through which insight on the regulation of gene expression in pathological tissue can be obtained. These studies have broad implications for understanding pathophysiological mechanisms of disease. Second, putative cell protective effects of heat shock proteins in brain tissue provide insight into biochemical mechanisms of selective neuronal vulnerability. These studies have extremely important clinical implications since cell sensitivity to injury can seemingly be modified. The role of heat shock proteins in hypoxic-ischemic brain injury is discussed forthwith.     

Sphingolipid activator proteins (SAPS) are stored together with glycosphingolipids in the infantile neuronal ceroid-lipofuscinosis (INCL)

The storage material isolated from the brains of patients with infantile neuronal ceroid-lipofuscinosis (INCL) contains, on average, 43% protein and 35% lipids on a dry weight basis. Recently we identified the major storage proteins as sphingolipid activator proteins (SAPs) A and D by direct sequencing. In the present study we used monospecific anti-sap-B-, anti-sap-C, and anti-sap-D-antisera in immunohistochemical and Western analyses to show that sap-D is, indeed, an integral component of the storage bodies. In contrast, no (or little) immunoreactivity for sap-B or sap-C was detected in the INCL storage granules. This observation is of interest for an understanding of the pathogenesis because the four SAPs are produced from a single precursor protein by proteolytic cleavage. Furthermore, we analysed the stored lipids on high performance thin layer chromatography combined with different staining techniques. In this preliminary analysis we found two glycosphingolipids, yet to be identified, to be common for all INCL patients. © 1995 Wiley-Liss, Inc.     

Immunohistochemical detection of bcl-2 protein in liver lesions: bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia

A proto-oncogene, bcl-2, encodes a protein that inhibits programmed cell death (apoptosis) and may play a role in cell and tissue differentiation. As bcl-2 appears to be involved in the turn-over of stem or precursor cells, it is thought to be operational in carcinogenesis pathways. However, apart from certain lymphomas, only limited data are available on the frequency of its expression in solid tumors. Immunohistochemical analysis with an antibody specific for bcl-2 protein was used to detect the protein in hepatocellular carcinomas and in one of the putative precursor lesions, liver cell dysplasia. We detected bcl-2 protein in 5 of 37 hepatocellular carcinomas. Immunoreactivity was not related to type, grade, or extent of PCNA staining of the tumours. No bcl-2 protein staining was observed in three types of liver cell dysplasia. Thus, bcl-2 is abnormally expressed in some hepatocellular carcinomas but not in potential tumour precursor cells.     

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.     

Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product

The complete nucleotide sequence of the Bunyamwera virus M RNA segment was determined from four overlapping cDNA clones and by primer extension. The RNA segment is 4458 bases in length, and encodes a single gene product in the viral complementary RNA. The predicted protein is 1433 amino acids long (mol wt 162,065), contains four potential glycosylation sites, and is relatively cysteine rich. It is presumed that the three proteins G1, G2, and NSM which have been mapped to the M RNA segment are synthesized as a precursor polyprotein which is subsequently proteolytically cleaved. A putative hydrophobic signal sequence at the amino terminus and a hydrophobic anchor sequence at the carboxy terminus of the predicted protein have been identified, in addition to internal regions of hydrophobicity of unknown function. The nucleotide and amino acid sequences of the Bunyamwera virus M segment have been compared with those of the snowshoe hare virus M segment (Y. Eshita and D. H. L. Bishop, Virology 137, 227-240, 1984). Common features include the overall architecture of the RNAs, single cysteine-rich primary gene products, and conservation of hydrophobic domains in the gene products. When aligned the amino acid sequences are 43% homologous, and 66 of 70 cysteine residues can be matched. The evolutionary significance of these findings is discussed.     

Gaucher's disease in the presence of normal glucocerebrosidase activity.

We encountered an infant with clinical and histopathologic features of Gaucher's disease (infantile, type 2) with normal glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, E.C.3.2.1.45) activity. Biochemical analysis was performed on leukocytes, cultured skin fibroblasts, and liver. Normal activity of glucocerebrosidase previously has been reported in an older child with juvenile onset (type 3) Gaucher's disease and attributed to a deficiency of a sphingolipid activator protein. These rare cases illustrate and expand our concept of Gaucher's disease and may have both diagnostic and therapeutic implications.     

A case of Krabbe's leukodystrophy without globoid cells

Krabbe's globoid cell leukodystrophy is a rare hereditary progressive neurological disease of infants, in which there is deficient activity of galactosylceramide beta-galactosidase. The pathological hallmark is the presence of multinucleated globoid cells in the white matter associated with severe myelin depletion and gliosis. We report a second case where galactosylceramide beta-galactosidase deficiency was proven but no globoid cells were found in the brain. Symptoms began within the first 10 months of life and a deficiency of galactosylceramide beta-galactosidase activity was demonstrated in peripheral blood leukocytes and skin fibroblasts. The child survived till 8 yrs 7 mths. The reason for the absence of globoid cells is not clear but may be related to different effects of the gene mutation on the four substrates or possibly the interaction of sphingolipid activator protein-2.     

Transthyretin-its function and pathogenesis

Transthyretin (TTR) is a transport protein for retinol-binding protein and thyroxin, and works as a rapid turnover protein. Recently, it has been used as a nutrition assessment protein in the assessment of the acute phase nutritional status in various diseases because it contains four tryptophans in the tetramer of the protein and its plasma half life is 1.9 days. However, the wild-type protein and its mutated form become a precursor protein of amyloid fibrils in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy(FAP), respectively. Recent biochemical and pathological studies revealed that instability of the terameric form of TTR by mutation and post-translational modifications leads to amyloid formation in the tissues of SSA and FAP. In the process of TTR amyloid formation, misfolding of TTR, the trigger of amyloid formation, is also induced. For these amyloid formation mechanisms, Cr3+ administration, BSB(FSB) therapy, gene therapy, and antibody therapy are now on-going therapeutic projects for FAP and SSA.     

Expression,regulation and function of human FcεRII (CD23) antigen

CD23, also known as the low affinity receptor for IgE (Fc epsilon RII), belongs to a novel superfamily of type-II integral membrane proteins. Fc epsilon RII expression was originally described on B cells but subsequent studies showed that CD23 is expressed on a variety of hematopoietic cells and is regulated by several cytokines (i.e., interleukin-4, interferons) in a tissue-specific manner. In some pathological conditions such as B-chronic lymphocytic leukemia, the CD23 gene is abnormally regulated resulting in CD23 overexpression. CD23 is not only an IgE receptor but also a membrane-bound precursor of soluble molecules that still bind IgE (sCD23 or IgE-binding factors). The functions of membrane CD23 are IgE-dependent and vary according to the cell types on which it is expressed. In contrast, sCD23, in addition to being an IgE regulatory molecule, displays multiple cytokine activities that are IgE-independent.     

Recent Advances in the Biochemistry of Sphingolipidoses

Glycosphingolipids are ubiquitous membrane components of eukaryotic cells. They participate in various cell recognition events and can regulate enzymes and receptors within the plasma membrane. Sphingolipidoses are due to an impaired lysosomal digestion of these substances. Glycosphingolipids are degraded by the action of exohydrolases, which are supported, in the case of glycosphingolipids with short oligosaccharide chains, by sphingolipid activator proteins. Five sphingolipid activator proteins are known so far, the GM2-activator and the SAPs, SAP-A to D (also called saposins). Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane derived glycosphingolipids into the lysosomes. Recent research focused on the topology of this process and on the mechanism and physiological function of sphingolipid activator proteins. Limited knowledge is available about enzymology and topology of glycosphingolipid biosynthesis. Recently, intermediates of this metabolic pathway have been identified as novel signalling molecules. Inhibition of glycosphingolipid biosynthesis has been shown to be beneficial in the animal model of Tay-Sachs disease. Mice with disrupted genes for lysosomal hydrolases and activator proteins are useful models for known human diseases and are valuable tools for the study of glycosphingolipid metabolism, the pathogenesis of sphingolipidoses and novel therapeutic approaches.     

The role of saposin C in Gaucher disease

Saposin C is one of four homologous proteins derived from sequential cleavage of the saposin precursor protein, prosaposin. It is an essential activator for glucocerebrosidase, the enzyme deficient in Gaucher disease. Gaucher disease is a rare autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene that exhibits vast phenotypic heterogeneity, despite its designation as a "simple" Mendelian disorder. The observed phenotypic variability has led to a search for disease modifiers that can alter the Gaucher phenotype. The PSAP gene encoding saposin C is a prime candidate modifier for Gaucher disease. In humans, saposin C deficiency due to mutations in PSAP results in a Gaucher-like phenotype, despite normal in vitro glucocerebrosidase activity. Saposin C deficiency has also been shown to modify phenotype in one mouse model of Gaucher disease. The role of saposin C as an activator required for normal glucocerebrosidase function, and the consequences of saposin C deficiency are described, and are being explored as potential modifying factors in patients with Gaucher disease.     

Characterization of development-related genes for the cestode<Emphasis Type="Italic"> Bothriocephalus acheilognathi</Emphasis>

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.     

Ligation of the bile duct and acute chloroform toxicity in the donkey

Donkeys with ligated bile ducts exhibited the biochemical, pathological and clinical features characteristic of the syndrome of hepatogenous photosensitization. Incoordination of movement, constipation, oedema, dermatitis and jaundice were accompanied by a rise in the concentration of total bilirubin and ammonia and a decrease in the level of total protein and calcium in serum.A single dose of chloroform given to donkeys by stomach tube produced centrilobular necrosis of the liver. There were neither skin lesions of photosensitization nor clinical jaundice.The effects of ligation of the bile duct and the administration of chloroform were additive. This was shown by the degree of damage of the liver and kidneys, the extent of changes in serum constituents and by the clinical features.