Characterization of a novel alpha-D-mannosidase from rat brain microsomes.

Because the availability and subcellular distribution of processing mannosidases in cells play such powerful roles in determining ultimate structures of glycoconjugates, we desired to identify, characterize, and investigate possible regulation of mannosidases in infected and noninfected lepidopteran insect cells. Since our previous observations that a mannosidase activity that converted Man6GlcNAc2 to Man5GlcNAc2 was enhanced in virus-infected cells, thus providing the necessary intermediate for further processing to complex-type oligosaccharides, we attempted purification of this enzyme. A mannosidase was isolated and purified from membranes, operationally defined as Golgi, of recombinant baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells. The molecular mass of this protein was approximately 63 kDa. Assays performed by measuring the conversion of NaB3H4-reduced Man6GlcNAc2-ol to Man5GlcNAc-[3H]GlcNAc2-ol demonstrated that the mannosidase activity was dependent on the presence of divalent cations, which was optimal for Ca2+ at pH 6.0. Inclusion of 1-deoxymannojirimycin resulted in 50% inhibition at a concentration of 20 microM, whereas swainsonine did not show such inhibition. No activity was observed with p-nitrophenyl alpha-D-mannoside (4 mM) as a substrate. The preferred reduced oligosaccharide substrate was Man6GlcNAc2-ol, with lower activities obtained with Man9GlcNAc2-ol, Man8GlcNAc2-ol, and Man7GlcNAc2-ol. With Man6GlcNAc2-ol as substrate, products smaller than reduced Man5GlcNAc2-ol were not observed. Mannose was also liberated from the glycoprotein, ovalbumin. These properties are consistent with an enzyme classification as a type I (alpha 1,2)-Man6-mannosidase.

Section: Discussionmentioning

confidence: 87%

mentioning

confidence: 99%

Purification and Properties of a Golgi-Derived (.alpha.1,2)-Mannosidase-I from Baculovirus-Infected Lepidopteran Insect Cells (IPLB-SF21AE) with Preferential Activity toward Mannose6-N-Acetylglucosamine2

Ren¹,

Bretthauer²,

Castellino³

1995

“…The hybrid-type sugar chains with the Manal-*-2Manal-*3Manal-*-6Man group found in the CD36 reported here do not meet with this biosynthetic pathway and suggest that the bovine mammary epithelial cells have a different processing pathway for asparagine-linked sugar chains which contains -mannosidases and an TV-acetylglucosaminyltransferase I with different substrate specificities. In support of this hypothesis, several -1,2-mannosidases which differ from the well known a-1,2-mannosidases in their substrate specificities, their sensitivities to inhibitors such as deoxymannojirimycin and swainsonine, and their kinetic and physical properties have been purified from a variety of animal tissues (Shoup & Touster, 1976;Tabas & Kornfeld, 1979;Forsee & Schutzbach, 1981;Tulsiani et al, 1982;Tulsiani & Touster, 1985;Bischoff & Kornfeld, 1986;Schweden et al, 1986;Forsee et al, 1989;Bonay & Hughes, 1991). These reports suggest that the trimming pathway of high mannose-type oligosaccharides to hybrid-type ones may be distinct in different cell types.…”

Section: Discussionmentioning

confidence: 87%

Structural study of the sugar chains of CD36 purified from bovine mammary epithelial cells: Occurrence of novel hybrid-type sugar chains containing the Neu5Ac.alpha.2.fwdarw.6GalNAc.beta.1.fwdarw.4GlcNAc and the Man.alpha.1.fwdarw.2Man.alpha.1.fwdarw.3Man.alpha.1.fwdarw.6Man groups

Nakata¹,

Furukawa²,

Greenwalt³

et al. 1993

CD36 is a glycoprotein included in the bovine milk fat globule membrane derived from mammary secretory epithelial cells during lactation. Asparagine-linked sugar chains were quantitatively released from CD36 as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by Bio-Gel P-4 column chromatography in combination with serial chromatography on immobilized lectin columns including a Wistaria floribunda agglutinin (WFA)-agarose column. WFA is known to bind oligosaccharides terminating with either an alpha- or beta-N-acetylgalactosamine residue. Structural studies of oligosaccharides in each fraction by sequential exoglycosidase digestion as well as methylation analysis revealed that CD36 contains high mannose-type, hybrid-type, and bi, tri-, and tetraantennary complex-type sugar chains. A portion of the hybrid-type and the complex-type sugar chains which bound to a WFA-agarose column (28% of all oligosaccharides) contained the GalNAc beta 1-->4GlcNAc group(s) instead of the Gal beta 1-->4GlcNAc group(s) in their outer chain moieties. Like oligosaccharides found in human luteinizing hormone [Weisshaar, G., Hiyama, J., Renwick, A. G., & Nimtz, M. (1991) Eur. J. Biochem. 195, 257-268], some of the GalNAc beta 1-->4GlcNAc groups found in the CD36 oligosaccharides were sialylated as the Neu5Ac alpha 2-->6GalNAc group. Furthermore, most of the hybrid-type sugar chains of CD36 with the Gal/GalNAc beta 1-->4GlcNAc beta 1-->2 outer chain on their Man alpha 1-->3 arm contained an unusual Man alpha 1-->2Man alpha 1-->3 group on their Man alpha 1-->6 arm.

“…This latter glycan has been shown (Lubas & Spiro, 1987) to be a different isomer from the Man8GlcNAc2 generated from the exo-ER -D-mannosidase cited above (Bischoff et al, 1986). This alternate processing route allows circumvention of the need for ER glucosidases I and II under certain conditions of glucosidase blockage. Finally, a rat brain microsomal «-Dmannosidase activity has been identified with a novel activity toward («1,2)-, («1,3)-, and («l,6)-linked Man residues and is capable of providing the core glycan, viz., Man(«l,3)-[Man(«l,6)]Man(|61,4)GlcNAc(|61,4)GlcNAc, which presumably is a substrate for GlcNAc transferase I (Tulsiani & Touster, 1985). Within the Golgi stack of some cells, there also appears to be a subcellular distribution of a-mannosidase I like enzymes.…”

Section: Discussionmentioning

confidence: 99%

.alpha.-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells

Davidson

Bretthauer²,

Castellino³

1991

Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS)