Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis.

106

Abstract. We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner al,3-1inked glucoses from Nlinked oligosaccharide precursor chains ofglycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisteruae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II. N-GLYCOSIDICALLY linked oligosaccharides of glycoproteins are transferred from a lipid-linked precursor, dolichol pyrophosphate-(N-acetylglucosamine)2-(mannose)9-(glucose)3, to specific asparagine residues on nascent polypeptides (22). This occurs in the lumen of the rough endoplasmic reticulum (RER)/ Posttranslational modifications of the oligosaccharide precursor chain start with the enzymatic removal of all three glucose residues by two aglucosidases (11,21,26,54,55). Glucosidase I cleaves off the terminal c~l,2-1inked glucose and glucosidase II the two inner a 1,3-1inked glucoses. Processing of the oligosaccharide to high mannose, hybrid, or complex forms continues by trimming 1,2-mannosidases. c~ 1,2-Mannosidase (mannosidase I) activities have been identified in ER (2) and Golgi apparatus (16,46,47,52). Therefore, at least one mannose residue is removed before glycoproteins enter the Golgi apparatus. Further conversion to complex type oligosaccharides occurs via the action of N-acetylglucosamine transferase I (46) and amannosidase II, both of which have been localized to Golgi apparatus by immunoelectron microscopy (10, 32).Though current data indicate that the early steps of oligosaccharide processing are compartmentalized, the precise intracellular location of glucosidase II is not known. Cellular fractionation studies have shown enrichment of glucosidase 11 activity (14) and immunoreactivity (4) in rough and smooth microsomal fractions. All three glucoses can be trimmed from G protein of vesicular stomatitis virus still associated with h Abbreviations used in thispaper: RER, rough endoplasmic reticulum; SER, smooth endoplasmic retieulum. ribosomes (1). Pulse chase studies show rapid removal of two glucoses from glycoprotein precursors (t,/2 <2 min, by glucosidase I, and 4/2 -5 min by glucosidase II), although the last is removed significantly slower than the others (after ~20-30 min, by glucosidase II) (21).It has been suggested that proteins of the ER enter parts of the Golgi apparatus (41). The ER proteins investigated so far do not contain posttranslational modifications attributable to passage through the Golgi apparatus (27,...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II.

Lucocq¹,

Brada²,

Roth³

1986

106

“…Glucosidase I removes rapidly (tt/2 <1 min) the terminal od,2-1inked glucose residue (Hubbard and Robbins, 1979). Glucosidase II hydrolyzes the two inner od,3-1inked glucose residues (Grinna and Robbins, 1979;Burns and Touster, 1982). The outer cd,3linked glucose residue is hydrolyzed more rapidly (t~ --5 rain) than the inner one (after •20-30 min) (Hubbard and Robbins, 1979).…”

mentioning

confidence: 99%

Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II.

Brada

Kerjaschki

Roth

1990

Abstract. Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II conmined species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.N '-glycosylated glycoproteins acquire their oligosaccharide chains by en bloc transfer of the lipid-linked precursor Glc3Man9GlcNAc2 to the nascent polypeptide. The oligosaccharide precursors are then modified by glycosidases and glycosyltransferases to generate high marmose-, hybrid-, or complex-type structures. The processing starts with the removal of glucose residues by glucosidases (Hirschberg and Snider, 1987). Glucosidase I removes rapidly (tt/2 <1 min) the terminal od,2-1inked glucose residue (Hubbard and Robbins, 1979). Glucosidase II hydrolyzes the two inner od,3-1inked glucose residues (Grinna and Robbins, 1979;Burns and Touster, 1982). The outer cd,3-linked glucose residue is hydrolyzed more rapidly (t~ --5 rain) than the inner one (after •20-30 min) (Hubbard and Robbins, 1979). In hepatocytes, glucosidase II is a high mannose-type glycoprotein and was localized to the rough and smooth endoplasmic reticulum by biochemical fractionation procedures (Grinna and Robbins, 1979;Brada and Dubach, 1984) and by immunoelectron microscopy (Lucocq et al., 1986). Golgi apparatus cisternae were free of immunolabel for glucosidase II although subcellular fractions of Golgi apparatus contained small amounts of the enzyme which almost certainly represented contamination by the endoplasmic reticulum. The data on the biosynthesis of glucosidase II obtained in a rat hepatoma cell line are in agreement with the above results (Strous et al., 1987). The enzyme subunit was found to be an endo H-sensitive, 100-kD glycoprotein that remained unchanged over long chase periods. The halflife of glucosidase II was not altered by monensin treatment, further supporting the notion that glucosidase II does not enter the Golgi apparatus in hepatocytes. Collectively, these data demonstrated that, in hepatocytes, glucosidase II is a resident glycoprotein of the endoplasmic reticulum which is retained therein by an unknown mechanism.In the present study we have investigated the distribution of glucosidase II in pig kidney. The examination of kidney tubular cells revealed unexpected findings. Our biochemical and immunocytochemical data ...

“…The profiles show the distribution of the ts045 G protein oligosaccharides before (A) and after (B) digestion with rat liver glucosidase II, and the pdTM12 G protein oligosaccharides before (C) and after (D) glucosidase II digestion. The arrows in A and C denote the elution position of high mannose oligosaccharides composed of ManTGlcNAc(7), MansGlcNAc(8), and MangGIcNAc(9).…”

mentioning

confidence: 99%

Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

Suh

Bergmann

Gabel

1989

Abstract. Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H] -mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78 % of the 3H-marmose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.T HE processing of asparagine-linked oligosaccharides from high mannose-type units to mature complextype structures occurs in a highly ordered series of reactions (21,27). Enzymes involved in the processing reactions reside within the ER and Golgi apparatus, and biochemical as well as morphological studies indicate that they are restricted to defined compartments along the secretory pathway (13,14,16,18,32,46,47). The physical location of an enzyme within the pathway generally corresponds with the temporal order in which it functions (21, 27). In a previous study (16) we took advantage of the ordered processing reactions to define the intracellular compartments that conmined mutant forms of the G protein of vesicular stomatitis virus (VSV). The mutant G proteins were synthesized in COS-1 cells after transfection with plasmids that coded for proteins whose carboxy termini possessed deletions or alterations. As a result of the alterations, the proteins accumulated at intracellular sites rather than at the cell surface (43,44). The structure of the oligosaccharides attached to the mutant G proteins reflected the site of accumulation within the cells. For example, the G protein encoded by A1473 was shown to accumulate in the ER by immunofluorescence (44), and its asparagine-linked oligosaccharides were processed to high mannose-type units containing eight mannose residues (16...