Golgi a-mannosidase II (a-Mll) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human a-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding a-MIl were isolated. One such gene was found to encode an isozyme, designated acMIIx. A 5-kb cDNA clone encoding a-MIIX was then isolated from a human melanoma cDNA library. However, comparison between a-MIIX and a-MIl cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while a-MI consists of 1144 amino acid residues. To reevaluate the sequence of a-MIIx cDNA, polymerase chain reaction (PCR) was-performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the a-MIIX genomic sequence revealed that alternative splicing of the a-MIIX transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of a-MIIX in various tissues, suggesting that the a-MIIX gene is a housekeeping gene. COS cells transfected with c-MIIX cDNA containing the full-length open reading frame showed an increase of a-mannosidase activity. The ca-MIIX gene was mapped to human chromosome 15q25, whereas the a-MIl gene was mapped to 5q21-22.a-Mannosidase (a-M) activities are involved in both biosynthesis and catabolism of N-linked glycans (1, 2). These enzyme activities are present in cells ranging from yeast to human. There are different forms of a-Ms: lysosomal a-Ms are soluble and involved in degradation of N-glycans, endoplasmic reticulum (ER) and Golgi a-Ms are involved in processing of newly synthesized N-glycans, and cytoplasmic a-Ms may be involved in degradation of dolichol intermediates that are not needed for protein glycosylation or oligosaccharides derived from glycoprotein turnover in the ER (1).. Substrate specificities of these a-Ms differ from each other, and Golgi a-MIl specifically hydrolyzes two peripheral mannosyl residues from Manal1-6(Manal --3)Manal ->6(GlcNAcf31--2Mana1 ->3)[Man/31 ->4GlcNAc,lB --4GlcNAc31 ->]asparagine structure. Several a-Ms have been cloned to date. These include Golgi a-MIT (3, 4), ER/cytosolic a-MI (5), two isozymes of Golgi a-MI (6-8), lysosomal a-M (9), Dictyostelium a-M (10), and yeast a-M (11) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

IL-1β is produced primarily by activated monocytes/macrophages. We report in this study that IL-1β induces the human pro-IL-1β (IL1B) gene promoter in human THP-1 monocytic cells. The −131 to +12 minimal IL1B promoter was induced by IL-1β in a dose-dependent manner. The promoter possesses two important transcription factor binding motifs, one for an ETS family transcription factor Spi-1 (PU.1), and the other a binding site for NF-IL6 (CCAAT/enhancer binding protein β). Autocrine promoter activity was completely inhibited by mutation of the Spi-1 site. Mutation of the NF-IL6 binding motif caused partial loss of activity. EMSAs using THP-1 cell nuclear extracts indicated that IL-1β significantly induced Spi-1 binding to its target site within the IL1B promoter that was maximal at 1 h after stimulation, correlating with the kinetics of IL-1β induction. The importance of Spi-1 was supported by our observation that Spi-1-deficient EL4 thymocytes exhibited IL-1β-induced activity only after transfection with a Spi-1 expression vector. Moreover, TNFR-associated factor 6 also required Spi-1 to activate the promoter. Transfection studies using Spi-1 mutant constructs showed that the TATA-binding protein binding and glutamine-rich domains of Spi-1 were important for IL-1β induction, whereas LPS induction required the proline, glutamic acid, serine, and threonine-rich domain containing serine 148 as well as the TATA-binding protein and glutamine-rich domains. We conclude that the IL1B promoter is an IL-1β-responsive sequence as a result of its ability to bind Spi-1 in response to IL-1β.

Keywords: a-mannosidase II; a-mannosidase IIx; Golgi; HPLC; N-glycan.The major biosynthetic pathway of N-glycans in higher organisms has been established [1]. In the endoplasmic reticulum (ER), a lipid-linked glucosylated high-mannose oligosaccharide is transferred from the dolichol donor to a protein acceptor. High-mannose oligosaccharides are then processed by a-glucosidases and a-1,2 mannosidases (MI) to produce Man 5 GlcNAc 2 (M 5 Gn 2 ). A key conversion of this oligosaccharide to complex-type oligosaccharides occurs in the medial Golgi, where N-acetylglucosaminyltransferase-I (GnT-I) adds N-acetylglucosamine to the Mana133Manb13 terminal of M 5 Gn 2 to form Gn 1 M 5 Gn 2 [2]. The Golgi a-mannosidase II (MII) then removes two mannosyl residues from Gn 1 M 5 Gn 2 to form Gn 1 M 3 Gn 2 [3], which are further modified to complex oligosaccharides by N-acetylglucosaminyltransferase-II (GnT-II), galactosyltransferase and sialyltransferases.The presence of an MII-related enzyme was suggested by studies on a human genetic disease, congenital dyserythropoietic anemia type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test). Although the primary gene defect of HEMPAS is heterogeneous [4±8], the biochemical phenotype is characterized as a failure in N-glycan processing. Furthermore, mutant mice in which the MII gene was inactivated by homologous recombination showed strikingly similar phenotypes to those exhibited by HEMPAS patients [9]. Although cells from MII-null mice completely lack MII activity and accumulate hybrid-type oligosaccharides, MII-null splenocytes and fibroblasts were found to synthesize complex-type oligosaccharides. This finding indicates that complex oligosaccharides can be synthesized by an MII-independent pathway, leading to the proposal of an alternative pathway and the prediction of an a-mannosidase, designated a-mannosidase III (MIII). MIII hydrolyzes two a-mannosyl residues in M 5 Gn 2 and produces M 3 Gn 2 , which is further converted to Gn 1 M 3 Gn 2 by GnT-I. Bonay and Hughes [10] found a novel a-mannosidase in rat liver. This enzyme, in a cobalt iondependent manner, hydrolyzed a132, a133, and a136 mannosyl linkages of N-glycans. As the MIII activity was not observed in the tissues of the MII-null mouse until cobalt was added, MIII is most certainly the same activity Eur.

The human T-cell leukemia virus type I (HTLV-I), which infects a wide variety of mammalian cells including monocytes and macrophages, encodes a transactivating protein designated as Tax. We now report that Tax induces the human prointerleukin-1β (IL1B) gene promoter in monocytic cells. In our transient transfection assays using human THP-1 monocytic cells, a chloramphenicol acetyltransferase (CAT) construct containing the IL1B promoter sequence between positions −131 and +12 showed an approximately 90-fold increase in activity following cotransfection of a Tax expression vector. Moreover, Tax synergized with lipopolysaccharide (LPS) to induce the IL1B promoter activity. Analyses of specific nucleotide substitutions further indicated that the Tax-induced transcriptional activation requires two transcription factor binding motifs within the IL1B promoter; one is a binding site for nuclear factor (NF)-IL6 (CCAAT/enhancer binding protein β, C/EBPβ), which belongs to the basic region-leucine zipper (bZIP) family and the other for Spi-1 (PU.1), which is an Ets family protein found principally in monocytes, macrophages, and B lymphocytes. In electrophoretic mobility shift assays (EMSA) using in vivo THP-1 nuclear extracts, Tax expression in THP-1 monocytic cells significantly increased binding of the two factors to their target IL1B promoter sequences. However, in contrast to NF-IL6 and Spi-1, DNA binding activity of Oct-1, an ubiquitously expressed octamer-binding protein was not affected by Tax. Additional EMSA using in vitro translated proteins also showed that recombinant Tax enhances DNA binding of both of recombinant NF-IL6 and Spi-1 proteins. These data were supported by our glutathione S-transferase (GST)-pulldown data, which indicated that Tax physically interacts with the two proteins. Based on the results obtained from the present study, we conclude that the IL1B promoter is a Tax-responsive sequence as a result of ability of Tax to induce binding of NF-IL6 and Spi-1 to the IL1B promoter sequence through protein-protein interaction.