Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum. Evidence for an unusual acid-stable phosphodiester.

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Abstract. A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, o~-mannosidase and /3-glucosidase. A few hours into the chase period, HMW570 had secreted 95 % of its radiolabeled ot-mannosidase and 86% of its radiolabeled ~-glucosidase as precursor polypeptides compared to the secretion of <10% of these forms from wild-type cells. Neither a-mannosidase nor/$-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both a-mannosidase and/~-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the a-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominately located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Section: Analysis Of Oligosaccharidesmentioning

confidence: 99%

mentioning

confidence: 99%

A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

Ebert

Freeze

Richardson

et al. 1989

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“…The above data indicate that the ~20,000 daltons of carbohydrate added cotranslationally to the cellular precursor polypeptide is completely removed by endo H treatment, while the oligosaccharides are much more resistant on the secreted precursor. The mechanism of endo H resistance in Dictyostelium enzymes appears to be the presence ofesterified sulfate on the N-linked mannose-rich oligosaccharide side chains rather than further carbohydrate modifications producing more complex structures (17). These molecules also contain variable amounts of esterified phosphate (17), which, in combination with the sulfate, contributes to the highly acidic character of the enzymes.…”

Section: Differences Between Cellular and Secreted Precursorsmentioning

confidence: 99%

“…The mechanism of endo H resistance in Dictyostelium enzymes appears to be the presence ofesterified sulfate on the N-linked mannose-rich oligosaccharide side chains rather than further carbohydrate modifications producing more complex structures (17). These molecules also contain variable amounts of esterified phosphate (17), which, in combination with the sulfate, contributes to the highly acidic character of the enzymes. Accordingly, differences in charge between the cellular and secreted precursors would suggest differences in their degree of sulfation and/or phos-phorylation.…”

Section: Differences Between Cellular and Secreted Precursorsmentioning

confidence: 99%

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum.

Mierendorf¹,

Cardelli²,

Dimond³

1985

In Dictyostelium discoideum, the lysosomal enzyme a-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (tt/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with Iysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.

“…Both subunit types are derived from a single precursor polypeptide (Mr 140,000) (34,39). Like a number of other lysosomal enzymes, ct-mannosidase contains N-linked carbohydrates which are modified in the Golgi apparatus by addition of phosphate and sulfate residues at the 6-position of mannoses (17)(18)(19). To better characterize the relationship between PSVs and lysosomes, we performed [35S]methionine and [35S]sulfate labeling experiments to study the origin of PSV-associated c~-mannosidase and bulk glycoproteins, respectively.…”

mentioning

confidence: 99%

Developing Dictyostelium cells contain the lysosomal enzyme alpha-mannosidase in a secretory granule.

Lenhard

Siegel

Free

1989

Abstract. The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal/galNAc polysaccharides from prespore cells of Dictyostelium.By combining the techniques of protein A-gold immunocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme t~-mannosidase with gal/galNAc polysaccharides in prespore vesicles and the spore coat. To determine the origin of prespore vesicles a series of pulsechase experiments were performed. Cells were labeled with [35S]methionine or [35S]sulfate at different times during development and allowed to differentiate in the presence of unlabeled methionine or sulfate for various periods of time. The cells were homogenized and intracellular organelles were separated using Percoll density gradient centrifugation. The distribution of [35S]methionine-labeled t~-mannosidase and [35S]sulfate-labeled glycoproteins in the Percoll gradients was determined. It was found that prespore vesicles contained protein which was previously found in lysosomes. Newly labeled protein also entered these vesicles. The data suggest that developing Dictyostelium cells either restructure preexisting lysosomes into prespore vesicles or transport protein between these two organeUes. We propose that secretory granules and lysosomes may have a common biosynthetic origin and may be evolutionarily related. a result of studies performed on a variety of cell types, it is now becoming evident that common events are involved in mediating secretion by lysosomes and secretory granules (for reviews on lysosomes and secretory granules see references 4, 25, and 42). Polymorphonuclear leukocytes and neutrophils secrete acid hydrolases from lysosomes as a consequence of increased phosphoinositide metabolism (8,28,41). Under these same conditions chromarlin cells secrete catecholamines from chromaffin granules (13, 50). Increases in intracellular inositol triphosphate, 1,2-diacylglycerol, calcium, and protein kinase C activity have been implicated to be important in mediating these secretion events. Such similarities between different organellar types suggests there may be a common evolutionary and biosynthetic origin for lysosomes and secretory granules.The prespore vesicle (PSV) ~ is a secretory granule which arises as Dictyostelium cells differentiate into spores. It conrains spore coat proteins (9, 10) and gal/galNAc polysaccharides (10, 24) which are exocytosed during the terminal stages of prespore cell differentiation. These vesicles were originally defined morphologically as having an electron-dense layer juxtaposed to the membrane and a fibrous matrix of variable appearance (21,22,32). We recently identified two distinct lysosomal enzyme-J. M. Lenhard's present address is