Nathan N. Aronson

Galactosyl transferase — The liver plasma membrane binding-site for asialo-glycoproteins

Abstract Agalacto-fetuin inhibits the binding of 125 I-asialo-fetuin by liver plasma membrane fragments. The chemically prepared agalacto-glycoprotein derivative is not a substrate for plasma membrane sialyl transferase and therefore this indicates that agalacto-fetuin is a true inhibitor of the membrane binding of 125 I-asialo-fetuin. The plasma membrane fraction also contains galactosyl transferase activity and the binding of 125 I-asialo-fetuin by plasma membranes is prevented by α-lactalbumin, a known inhibitor of glycoprotein-galactosyl transferase. These data indicate that galactosyl transferase is the liver plasma membrane component which binds asialo-glycoproteins.

Cloning and sequence analysis of a cDNA for human glycosylasparaginase. A single gene encodes the subunits of this lysosomal amidase.

We have isolated a full‐length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221‐bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the α and β subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post‐translationally processed to generate two subunits of approx. 19.5 (α) and 15 (β) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the α and β subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the α and β subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (α) and 15 (β) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the α and β subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.

Post-translational processing and Thr-206 are required for glycosylasparaginase activity

Lysosomal glycosylasparaginase is encoded as a 36.5 kDa polypeptide that is post‐translationally processed to subunits of 19.5 kDa (heavy) and 15 kDa (light). Recombinant glycosylasparaginase has been expressed in Spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. Only the subunit conformation was functional indicating glyeosylasparaginase is encoded as an inactive zymogen. The newly created amino terminal residue of the light subunit following maturation, Thr‐206, is believed to be involved in the catalytic mechanism [1992, J. Biol. Chem. 267,6855‐6858]. Here we have constructed two amino acid substitution mutants replacing Thr‐206 with Ala‐206 or Ser‐206 and demonstrate that both destroy enzyme activity.

Effects of glucagon and insulin on liver lysosomes.

Abstract Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through autophagy , whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The t 1 2 for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.

Localization of an RNA-degrading system of enzymes on the rat liver plasma membrane

Abstract In comparison to other subcellular fractions, isolated rat liver plasma membrane has been shown to have a high capacity to degrade RNA at pH 8.8. 1. 1. Upon incubation of RNA with these membranes 0.056 μmoles of acid-soluble products and 0.023 μmole of inorganic phosphate are released per min per mg of membrane protein. This rate of RNA hydrolysis is comparable to that catalyzed by rat liver lysosomes. 2. 2. The activities of four enzymes, phosphodiesterase I (EC 3.1.4.1), 3′-nucleotidase (EC 3.1.3.6), 5′-nucleotidase (EC 3.1.3.5) and an endonuclease, are involved in this degradation of RNA. 3. 3. All four of these activities are localized with the plasma membrane fraction of liver. 4. 4. When phosphodiesterase I and 3′- and 5′-nucleotidases are inhibited by the the addition of EDTA, the hydrolysis of RNA still occurs. This implies that another enzyme capable of degrading RNA, yet not inhibited by EDTA, must be present on liver plasma membrane. 5. 5. When EDTA is absent from the reaction medium a large quantity of nucleosides is produced, but when EDTA is present, no significant amount of nucleosides or mononucleotides is released. This indicates that the non-EDTA inhibited activity is endonuclease. 6. 6. The pH optimum of the endonuclease is 7.7, while the optimum of overall RNA hydrolysis by plasma membrane is 9.3.

Genomic structure of human lysosomal glycosylasparaginase

The gene structure of the human lysosomal enzyme glycosylasparaginase was determined. The gene spans 13 kb and consists of 9 exons. Both 5′ and 3′ untranslated regions of the gene are uninterrupted by introns. A number of transcriptional elements were identified in the 5′ upstream sequence that includes two putative CAAT boxes followed by TATA‐like sequences together with two AP‐2 binding sites and one for Sp1. A 100 bp CpG island and several ETF binding sites were also found. Additional AP‐2 and Sp1 binding sites are present in the first intron. Two polyadenylation sites are present and appear to be functional. The major known glycosylasparaginase gene defect G488→C, which causes the lysosomal storage disease aspartylglycosaminuria (AGU) in Finland, is located in exon 4. Exon 5 encodes the post‐translational cleavage site for the formation of the mature α/β subunits of the enzyme as well as a recently proposed active site threonine, Thr206.

Uptake of asialo-glycophorin by the perfused rat liver and isolated hepatocytes.

Perfused rat livers took up asialo-glycophorin, a glycoprotein derived from human erythrocyte membranes, with a t1/2 for clearance of 7 min. As a comparison, asialo-orosomucoid was taken up by this system with a t1/2 of 3.5 min. Both proiteins were digested and their 125I labels were released to the perfusate as free 125I-. EGTA completely inhibited uptake of these glycoproteins, but not uptake of denatured bovine serum albumin. Addition of Ca2+ reversed the inhibition nearly completely. Isolated hepatocytes had an uptake rate of approximately 3 ng/min per 10(6) cells for the asialo forms of glycophorin, orosomucoid and fetuin. Cellular uptake of each of these asialoglycoproteins could be inhibited by one of the other proteins. Asialo-fetuin caused a 95% inhibition of the uptake rate of asialo-orosomucoid by the perfused liver. This fetal calf glycoprotein had a similar inhibitory effect on asialo-glycophorin, but only after an initial 40% of the asialo-glycophorin had been taken up by the liver at an almost normal rate during the first 30 min of perfsuion. The possibility of an alternative hepatic removal system for asialo-glycophorin is suggested.

Deletion of exon 8 causes glycosylasparaginase deficiency in an African American aspartylglucosaminuria (AGU) patient

We have indentified a T‐to‐ T transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU) patient, This mutation causes abnormal splicing of glycosylasparaginase pre‐mRNA by joining exon 7 to 9 and excluding 134 bp exon 8. The effect of the mutation is compounded by a frame shift that occurs after the deletion site resulting in premature translational termination. The truncated AGU protein was neither catalytically active nor processed into mature α and β subunits. Both this and a previously characterized Finnish AGU mutation appear to affect folding of the single‐chain precursor of glycosylasparaginase and thereby prevent transport of the enzyme to lysosomes.

Stereospecific synthesis of an α-mannosidase inhibitor relatedto swainsonine

Abstract A stereospecific synthesis of pyrrolidine 2 , an analog of swainsonine and an inhibitor of lysosomal α-D-mannosidase, is described.

Tissue locations for the turnover of radioactively labeled rat orosomucoid in vivo

Tissues involved in the turnover of rat serum orosomucoid were identified by methods designed to cause lysosomal trapping of radiolabel at the sites of glycoprotein degradation. 125I-, [3H]Raffinose-, and [1-14C]glucosamine-labeled orosomucoid exhibited serum half-lives of 20, 20, and 27 h when injected intravenously into rats. As expected, the asialo derivative of [3H]raffinose-labeled rat orosomucoid was lost very rapidly from the circulation and recovered quantitatively in the liver within 30 min. At 50 h after injection of [3H]raffinose-asialo-orosomucoid the liver retained 38% of the radioactivity while the remainder was found in the gastrointestinal tract and urine. Chromatography of the urine on Bio-Gel P-4 revealed a single radioactive product that eluted similar to raffinose-lysine. The same material was found in the liver. This ability of the [3H]raffinose label to resist metabolic disposal was used to evaluate tissue catabolism of native rat orosomucoid. Comparison of the tissue radioactivity in experiments using 125I- and [3H]raffinose-labeled derivatives of the nondesialylated glycoprotein showed kidney, liver, and muscle to be most active in 3H accumulation. However, the [3H]raffinose metabolites excreted in the urine was markedly different from those produced from asialo-orosomucoid and in contrast there was minimal loss of label to the gastrointestinal tract from the native substrate. Leupeptin, an inhibitor of lysosomal thiol cathespins, was administered continuously to rats by a subcutaneous osmotic pump. At 24 h after injection of 125I-orosomucoid, leupeptin-treated rats showed a net 16% increase in tissue radioactivity above sham-operated animals and a corresponding decrease occurred in the radioactivity associated with the gastrointestinal tract and urine. Tissues that exhibited increases in radioactivity were kidney, muscle, liver, and hide. The different behavior of labeled native and asialo-orosomucoids suggests that the hepatic galactose receptor system plays, at most, a limited role in maintaining homeostasis of the native glycoprotein.