Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency

Summary Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.Abbreviations PD Pseudo arylsulfatase-A Deficiency - ARA Arylsulfatase-A     

Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.     

Elevation of superior vena caval pressure increases extravascular lung water after endotoxemia

In many sheep Escherichia coli endotoxin results in pulmonary hypertension, increased microvascular permeability, pulmonary edema, and increased central venous pressure. Since lung lymph drains into the systemic veins, increases in venous pressure may impair lymph flow sufficiently to enhance the accumulation of extravascular fluid. We tested the hypothesis that, following endotoxin, elevating the venous pressure would increase extravascular fluid. Thirteen sheep were chronically instrumented with catheters to monitor left atrial pressure (LAP), pulmonary arterial pressure (PAP), and superior vena caval pressure (SVCP) as well as balloons to elevate LAP and SVCP. These sheep received 4 micrograms/kg endotoxin, and following the pulmonary hypertensive spike the left atrial balloon was inflated so that (PAP + LAP)/2 = colloid osmotic pressure. It was necessary to control PAP + LAP in this way to minimize the sheep-to-sheep differences in the pulmonary hypertension. We elevated the SVCP to 10 or 17 mmHg or allowed it to stay low (3.2 mmHg). After a 3-h period, we killed the sheep and removed the right lungs for determination of the extravascular fluid-to-blood-free dry weight ratio (EVF). Sheep with SVCP elevated to 10 or 17 mmHg had significant increases in EVF (5.2 +/- 0.1 and 5.6 +/- 1.2) compared with the sheep in which we did not elevate SVCP (EVF = 4.5 +/- 0.4). These results indicate that sustained elevation in central venous pressure in patients contributes to the amount of pulmonary edema associated with endotoxemia.     

EVIDENCE FOR THE OCCURRENCE AND DISTRIBUTION OF INORGANIC POLYPHOSPHATES IN VERTEBRATE TISSUES

Evidence for the occurrence of polyphosphates having apparent chain-lengths ranging from less than 10 to over 5000 orthophosphate units has been found in adult brain as well as in a number of other mammalian tissues which have been examined. There appears to be three times as much polyphosphate in rat brain as there is in rat liver. Adult rat brain appears to contain at least 15 μg of phosphorus as polyphosphate per g of fresh tissue. In addition, neural polyphosphate is extremely labile to catabolic degradation after death whereas hepatic polyphosphate is relatively more stable. The nature of this inorganic polymer was elucidated through use of the technique of ³¹P nuclear magnetic resonance spectroscopy. Further structural evidence was obtained by application of the isotopic dilution technique to ³²P-labelled neural polyphosphate mixed with an abiotically prepared inorganic polyphosphate. The conditions and rates of hydrolysis of the biological polyphosphate and a non-biological polyphosphate were comparable.