Isolation and Characterization of a Class II A-Mannosidase Cdna from Lepidopteran Insect Cells

Glycobiology vol. 7 no. 1 pp. 113-127, 1997 Isolation and characterization of a class II a-mannosidase cDNA from lepidopteran insect cells Donald LJarvis2, Dwight A.Bohlmeyer, Yung-Feng Introduction Liao1, Kristen K.Lomax, Roberta K.Merkle1, Carla 1 Weinkauf and Kelley W.Moremen Lepidopteran insect cells are used widely as hosts for foreign Department of Entomology and Center for Advanced Invertebrate Molecular gene expression by recombinant baculoviruses and, like bac- Sciences, Texas A&M University, College Station, TX 77843, USA and terial expression systems, the insect cell-baculovirus system 'Department of Biochemistry and Molecular Biology and Complex can provide high levels of foreign gene expression (Summers Carbohydrate Research Center, University of Georgia, Athens, GA 30602, and Smith, 1987; Miller, 1988; O'Reilly et al., 1992). The USA Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 insect ceU-baculovirus system also has eukaryotic protein pro- ^o whom correspondence should be addressed cessing capabilities and, therefore, is one of the best tools Lepidopteran insect cells are used routinely as hosts for currently available for foreign glycoprotein production. How- foreign glycoprotein expression by recombinant baculovi- ever, the nature of the N-glycosylation pathway in lepidopteran ruses, but the precise nature of their N-glycosylation path- insect cells and, particularly, in baculovirus-infected lepidop- way remains poorly defined. These cells clearly have pro- teran insect cells, remains poorly defined. cessing glucosidases and mannosidases that can convert By comparison, the N-glycosylation pathway in higher eu- caryotes is well-defined (reviewed by Kornfeld and Kornfeld, precursors to Man3GlcNAc2 structures and fucosyltrans- ferases that can add fucose to the oligosaccharide core. 1985). This pathway begins with the cotranslational transfer of However, their ability to extend these structures to produce preformed GlCjMangGlcNAcj oligosaccharides from a doli- complex side chains like those found in mammalian cells chol-linked precursor to nascent polypeptide chains on the lu- remains to be determined. To begin to examine this path- minal face of the endoplasmic reticulum (ER), followed by way at the molecular genetic level, we isolated and charac- immediate trimming of glucose residues within the ER. Addi- terized a class II ot-mannosidase (a-mannosidase II) cDNA tional trimming of glucose residues and as many as three of the from Sf9, a lepidopteran insect cell line. In mammalian nine mannose residues can occur within the ER. As glycopro- cells, this enzyme catalyzes the committed step in the path- teins transit the Golgi complex, the last a-l,2-linked mannose way converting N-linked carbohydrates to complex forms. residues are removed by 'class F ot-mannosidases, producing a Degenerate primers against conserved regions in known Man5GlcNAc2 structure (Moremen et al., 1994). Following the class II a-mannosidase protein sequences were used to gen- addition of a single GlcNAc residue by GlcNAc transferase I, erate an a-mannosidase H-specific PCR product from Sf9 two more mannose residues are removed by Golgi a-manno- cell DNA. Sequence information from this product was sidase II, a 'class II' mannosidase (Moremen et al, 1994). This used to isolate a partial cDNA clone, the 5' end was isolated is the committed step in the synthesis of complex type oligo- by ligation-anchored PCR, and the full length a-mannosi- saccharides, which are subsequently produced by Golgi glyco- dase II cDNA was assembled. This cDNA contained a long syltransferases that extend the trimmed structures by adding open reading frame predicted to encode an 1130 amino acid N-acetylglucosamine, galactose, fucose, and sialic acid resi- protein with 37% identity to human Golgi a-mannosidase dues (reviewed by Komfeld and Komfeld, 1985; Paulson and II and with a type II membrane topology, a feature of all Colley, 1989; Moremen et al., 1994). known Golgi processing enzymes. Southern blotting indi- Most information on the N-glycosylation pathway in lepi- cated that a-mannosidase II is a single copy gene in Sf9 dopteran insect cells has come from structural studies on for- cells. Other Lepidoptera had related a-mannosidase II eign glycoproteins expressed in baculovirus-infected cell lines genes, but there was variation among different genera, and or in larvae (reviewed by Jarvis and Summers, 1992; O'Reilly the Sf9 a-mannosidase II cDNA did not cross-hybridize et al., 1992; Jarvis, 1993a). These studies have demonstrated with DNA from animals outside Lepidoptera. Steady-state that lepidopteran insect cells have processing glucosidases and levels of a-mannosidase II RNA were low in uninfected Sf9 mannosidases which convert high mannose oligosaccharides to cells and even lower after baculovirus infection. The trimmed structures with as few as three mannose residues in v#ro-translated Sf9 a-mannosidase II protein had the (pauci-mannose structures). Several lines of evidence indicate expected size and was translocated and N-glycosylated by that these cells also have a fucosyltransferase that can add microsomal membranes. Expression of the Sf9 a-mannosi- fucose to the core Asn-linked GlcNAc residue (Staudacher et dase II cDNA in the baculovirus system produced large al., 1992), but it remains unclear whether insect cells have the amounts of a protein with the expected size and swainso- ability to extend these trimmed structures, as in mammalian nine-sensitive a-mannosidase II activity towards an aryl- cells. Most structural data indicate that baculovirus-expressed a-mannoside substrate. These results demonstrate that Sf9 glycoproteins (Kuroda et al., 1990; Chen and Bahl, 1991; Chen cells encode and express an a-mannosidase II with prop- etal., 1991; Wathen etal, 1991; Hogeland et al., 1992; Knep- erties similar to those of the mammalian enzyme. per et al., 1992; Grabenhorst et al., 1993; Yeh et al., 1993; Manneberg et al., 1994; Jarvis and Finn, 1995) and native Key words: class II ot-mannosidase/cDNA/lepidopteran insect insect glycoproteins (Butters et al, 1981; Hsieh and Robbins, cells/baculovirus expression system 1984; Ryan et al., 1985; Nagao et al., 1987; Williams et al., 1991) have only trimmed and fucosylated high mannose or © Oxford University Press 113 D.LJarvis et at pauci-mannose oligosaccharides. However, some recent stud- human a-mannosidase II (42.3% and 43.3% identity, respec- ies indicate that lepidopteran insect cell lines have GlcNAc tively) and D .discoideum and human lysosomal a-mannosi- transferase I and II activities (Altmann et al., 1993; Velardo et dase (29.4% and 26.1% identity, respectively), were identified al., 1993) and that they can produce glycoproteins with termi- by diis analysis. This suggested that the Sf9 amplimer was nal GlcNAc (Kubelka et al., 1994; Ackermann et al., 1995), derived from a gene that is related to the class II mannosidases Gal (Ogonah et al., 1996), and sialic acid (Davidson et al, and is more similar to the Golgi processing than the lysosomal 1990; Davidson and Castellino, 1991a). mannosidases. While structural biochemistry has provided much useful in- The Sf9 amplimer DNA sequence was used to design exact- formation on the N-glycosylation pathway in lepidopteran in- match primers against the putative Sf9 a-mannosidase II cod- sect cells, this approach is limited because it is indirect and ing region, and these primers were used for PCRs with total \ provides only a retrospective view of the processing pathway, DNA from an unfractionated Sf9 cDNA library, as described in which must be inferred from the structures of the end-products. Materials and methods. Electrophoretic analysis of the reaction Conclusions about the processing pathway can be complicated products revealed one major DNA fragment of about the same by degradative pathways, which might alter the product of the size as the RT-PCR product (data not shown). The same result biosynthetic pathway and lead to misinterpretations (Licari et was obtained with total A. DNA from an unfractionated Sf9 al., 1993; Jarvis and Finn, 1995). Finally, conclusions based on genomic DNA library or with the pCRII clone containing the Downloaded from https://academic.oup.com/glycob/article/7/1/113/725524 by guest on 01 October 2021 structural data from any one glycoprotein might apply only to Sf9 amplimer, but not in negative controls which lacked tem- that glycoprotein and not to the pathway in general. An alter- plate DNA or contained one specific and one nonspecific native approach that circumvents these problems is to use mo- primer (data not shown). These results indicated that the un- lecular genetics to isolate genes encoding glycoprotein pro- infected Sf9 cell cDNA library included a clone containing the cessing enzymes. This makes it possible to study these genes, putative Sf9 a-mannosidase II coding region, prompting us to their expression, and, ultimately, the properties of the enzymes proceed to isolate this cDNA by using a sibling selection and they encode. A large number of cDNAs encoding various gly- PCR screening approach (Moremen, 1989). Three positive coprotein processing enzymes have been cloned and charac- clones were isolated through sibling selection and three rounds terized in other systems (reviewed by Paulson and Colley, of plaque purification, as described in Materials and methods 1989; Lowe, 1991; Joziasse, 1992; Moremen etai, 1994; Field (data not shown). The cDNA inserts were excised

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