Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.

Insect cells encode a class II alpha-mannosidase with unique properties.

Previously, we cloned and characterized an insect (Sf9) cell cDNA encoding a class II alpha-mannosidase with amino acid sequence and biochemical similarities to mammalian Golgi alpha-mannosidase II. Since then, it has been demonstrated that other mammalian class II alpha-mannosidases can participate in N-glycan processing. Thus, the present study was performed to evaluate the catalytic properties of the Sf9 class II alpha-mannosidase and to more clearly determine its relationship to mammalian Golgi alpha-mannosidase II. The results showed that the Sf9 enzyme is cobalt-dependent and can hydrolyze Man(5)GlcNAc(2) to Man(3)GlcNAc(2), but it cannot hydrolyze GlcNAcMan(5)GlcNAc(2). These data establish that the Sf9 enzyme is distinct from Golgi alpha-mannosidase II. This enzyme is not a lysosomal alpha-mannosidase because it is not active at acidic pH and it is localized in the Golgi apparatus. In fact, its sensitivity to swainsonine distinguishes the Sf9 enzyme from all other known mammalian class II alpha-mannosidases that can hydrolyze Man(5)GlcNAc(2). Based on these properties, we designated this enzyme Sf9 alpha-mannosidase III and concluded that it probably provides an alternate N-glycan processing pathway in Sf9 cells.

Human homologs of the Xenopus oocyte cortical granule lectin XL35.

The cDNAs encoding two human homologs of the Xenopus oocyte lectin, XL35, were isolated from a small intestine cDNA library and termed HL-1 and HL-2. The deduced amino acid sequence of each homolog is about 60% identical and 80% similar to that of XL35, and none of these sequences contains the C-type lectin motif, although it is known that XL35 requires calcium for ligand binding. By Northern analysis, HL-1 transcripts are present at relatively high levels in heart, small intestine, colon, thymus, ovary, and testis. HL-2 transcripts, by contrast, are expressed only in small intestine. Immunocytochemistry using a polyclonal antibody produced against XL35 shows HL-1 protein to be localized exclusively in endothelial cells in colon, thymus, liver, and other tissues. Primary cultures of human aortic endothelial cells are positive for HL-1 expression by immunoblotting and by PCR analysis, but several other human cell types are not. HL-1 and -2 are both encoded at chromosome 1q23, the same locus that encodes the selectins. XL35, HL-1 and -2, and another mouse homolog are members of a new family of proteins whose members most likely perform diverse functions.

Molecular cloning and expression of an alpha-mannosidase gene in Mycobacterium tuberculosis.

Mannose is a major component of glycolipids and glycoproteins of the cell envelope of M. tuberculosis (Mtb). However, the enzymes involved in the biosynthesis and catabolism of mannosylated glycans are largely unknown. We demonstrate alpha-mannosidase activity towards the fluorescent substrate 4-methylumberlliferyl-alpha-D-mannopyranoside (4MU-Man) in cell lysates of attenuated and virulent Mtb bacilli, with two-fold higher activity in the virulent strain Erdman. Mannosidase activity was optimal at pH 6.5, was not inhibited by deoxymannojirimycin (dMNJ), was mildly inhibited by swainsonine (SW) and stimulated two-fold by EDTA. GenBank BLAST analysis for sequences homologous to eukaryotic alpha-mannosidases revealed a 3.6 kb putative gene (Rv0648) in Mtb cosmid SCY20H10 (Acc# z92772), with strong homology (48%) to the rat ER/cytosolic alpha-mannosidase and containing signature sequences of class 2 mannosidases. By RT-PCR, gene Rv0648 was found differentially expressed, with lower expression during growth in A549 pneumocyte cultures. Gene Rv0648 was cloned, expressed in E. coli, and alpha-mannosidase activity in cell lysates determined. Expression of alphaMan-pET in E. coli cells resulted in an eight-fold increase in mannosidase activity toward 4-MU-Man, upon IPTG induction. Partial purification of the histidine-tagged Mtb mannosidase by metal chelation affinity chromatography, and analysis by SDS-PAGE, showed a protein with the predicted m.w. of 137.5 kDa. Enzyme assays of the column fractions showed alpha-mannosidase activity toward synthetic aryl-mannose substrates, in fractions enriched in the recombinant Mtb mannosidase. These results demonstrate that gene Rv0648 encodes an active alpha-mannosidase in Mtb.

Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases.

Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases.

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.

Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis.

We have isolated a full-length cDNA clone encoding a human alpha1, 2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells. Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5'-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA. The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids). Northern blots detected a transcript of approximately 2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged full-length form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization. When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single alpha1,2-mannose residue from Man(9)GlcNAc(2) to produce a unique Man(8)GlcNAc(2) isomer (Man8B). The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca(2+). The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine. The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I. Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man(9)GlcNAc(2) oligosaccharides to Man(5)GlcNAc(2).

Isolation and characterization of the gene encoding the mouse broad specificity lysosomal alpha-mannosidase1.

A genomic clone encoding the mouse lysosomal alpha-mannosidases was isolated and the gene was found to be encoded by 24 exons spanning approximately 14.5 kb of genomic DNA. The intron-exon boundaries were conserved between the mouse, human, and bovine lysosomal alpha-mannosidase genes as well as being partially conserved in several other species. In order to define the promoter of the mouse mannosidase gene, >1 kb of DNA sequence was obtained upstream from the respective initiation codon. The transcription start site was identified by a 5'-RACE procedure and putative promoter elements were identified by expression of promoter/reporter constructs. Fluorescence in situ hybridization analysis using the mouse and human mannosidase genomic clones as probes, localized the mouse gene to chromosome 8, at band position 8C2, and the human gene to chromosome 19p13.2, a region syntenic to the lysosomal mannosidase gene on mouse chromosome 8.

Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1.

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.

Purification, crystallization and preliminary X-ray crystallographic analysis of recombinant murine Golgi mannosidase IA, a class I alpha-mannosidase involved in Asn-linked oligosaccharide maturation.

Golgi mannosidase IA is a class I alpha-mannosidase which catalyzes the conversion of Man9GlcNAc2 or Man8GlcNAc2 oligosaccharide substrates to Man5GlcNAc2 during the maturation of Asn-linked oligosaccharides. The enzyme is a type II membrane protein, and a recombinant form of mannosidase IA from mouse, lacking the transmembrane domain, has been expressed in Pichia pastoris, purified to homogeneity and crystallized by the hanging-drop vapor-diffusion method. The crystals grow as thin rods, with unit-cell dimensions a = 54.9, b = 135.01, c = 69.9 A. The crystals exhibit the symmetry of space group P2221 and diffract to 2.8 A resolution. The asymmetric unit contains one monomer ( approximately 53 kDa) and has a solvent content of 59%.

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha-mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.

Substrate specificities of recombinant murine Golgi alpha1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases.

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2-cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.

Human lysosomal and jack bean alpha-mannosidases are retaining glycosidases.

The stereochemical course of the hydrolyses catalysed by two alpha-mannosidases has been determined directly by 1H NMR. Synthetic substrates were incubated with the enzymes and the anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift of the anomeric proton at the hemiacetal centre. Both mannosidases were found to catalyse hydrolysis with retention of stereochemistry at the anomeric position. Human lysosomal alpha-mannosidase (a class II mannosidase) is a member of the glycosidase family 38 and thus has sequence similarity with several alpha-mannosidases responsible for glycoprotein biosynthesis. Jack bean alpha-mannosidase was shown to be mechanistically similar to the lysosomal enzyme and will provide a useful model system in mechanistic studies and inhibitor design.

Cloning, expression, purification, and characterization of the murine lysosomal acid alpha-mannosidase.

Catabolism of alpha-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal alpha-mannosidase (EC 3.2.1.24). Based on regions of protein sequence conservation between the lysosomal alpha-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing alpha 1,3/1,6-mannosidase, alpha-mannosidase II, we have cloned a cDNA encoding the murine lysosomal alpha-mannosidase. The longest of the clones was 3.1 kb in length and encoded a polypeptide of 992 amino acids containing a putative NH2-terminal signal sequence and 11 potential N-glycosylation sites. The deduced amino acid sequence was 76.5% identical to the human lysosomal alpha-mannosidase and 38.1% identical to the lysosomal alpha-mannosidase from D. discoideum. Expression of the cDNA in Pichia pastoris resulted in the secretion of an alpha-mannosidase activity into the culture medium. This recombinant expression product was purified and was shown to have enzymatic characteristics highly similar to the enzyme purified from mammalian sources and to the human lysosomal alpha-mannosidase cDNA expressed in Pichia. These characteristics include a similar pH optimum, Km, Vmax, inhibition by swainsonine, and activity toward natural substrates. Northern blots identified a major 3.5 kb RNA transcript in all murine tissues tested. A minor transcript of 5.4 kb was also detected in some murine tissues similar to the alternatively spliced transcripts that have been previously identified in human tissues.

Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis.

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development.

cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been isolated, characterized, and expressed in bacteria. This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope. A lectin with similar specificity has been purified by others from blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after fertilization in regulating cell adhesion and migration. We have used melibiose affinity chromatography to isolate the oocyte lectin (monomer molecular masses of about 45 and 43 kDa) and shown that after exhaustive treatment with N-glycanase, only one major protein band at 35 kDa was observed, suggesting that a single polypeptide with variable N-linked glycosylation is expressed in the oocyte. After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ovary lambda gt11 library encoding a protein of 313 amino acids with three potential N-linked oligosaccharide sites. Although this lectin, termed XL35, requires calcium ions for oligosaccharide binding, its sequence does not contain the sequence motif defined for "C-type" lectins. A 6-Histagged from of the lectin was expressed in E. coli and purified on a Ni(2+)-NTA column from bacterial extracts. The recombinant lectin was active using an agglutination assay, and this activity was inhibitable by EDTA and melibiose, properties exhibited by the native lectin. Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family. Northern blot analysis demonstrated that the lectin mRNA is expressed in oocytes and remains at relatively high levels through late gastrulae, continuing until tadpole stages. The persistence of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed and functions during morphogenesis.

Isolation and characterization of a class II alpha-mannosidase cDNA from lepidopteran insect cells.

Lepidopteran insect cells are used routinely as hosts for foreign glycoprotein expression by recombinant baculoviruses, but the precise nature of their N-glycosylation pathway remains poorly defined. These cells clearly have processing glucosidases and mannosidases that can convert precursors to Man3GlcNAc2 structures and fucosyltransferases that can add fucose to the oligosaccharide core. However, their ability to extend these structures to produce complex side chains like those found in mammalian cells remains to be determined. To begin to examine this pathway at the molecular genetic level, we isolated and characterized a class II alpha-mannosidase (alpha-mannosidase II) cDNA from Sf9, a lepidopteran insect cell line. In mammalian cells, this enzyme catalyzes the committed step in the pathway converting N-linked carbohydrates to complex forms. Degenerate primers against conserved regions in known class II alpha-mannosidase protein sequences were used to generate an alpha-mannosidase II-specific PCR product from Sf9 cell DNA. Sequence information from this product was used to isolate a partial cDNA clone, the 5' end was isolated by ligation-anchored PCR, and the full length alpha-mannosidase II cDNA was assembled. This cDNA contained a long open reading frame predicted to encode an 1130 amino acid protein with 37% identity to human Golgi alpha-mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that alpha-mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related alpha-mannosidase II genes, but there was variation among different genera, and the Sf9 alpha-mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of alpha-mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 alpha-mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 alpha-mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive alpha-mannosidase II activity towards an aryl-alpha-mannoside substrate. These results demonstrate that Sf9 cells encode and express an alpha-mannosidase II with properties similar to those of the mammalian enzyme.

Cloning, expression, purification, and characterization of the human broad specificity lysosomal acid alpha-mannosidase.

We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from alpha-mannosidosis fibroblasts, and p-nitrophenyl-alpha-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, Km, and Vmax characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No alpha-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb alpha-mannosidase cDNA immunoprecipitated lysosomal alpha-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including alpha-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal alpha-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.

Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

Golgi alpha-mannosidase II (alpha-MII) 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 alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22.

Remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyltransferase.

The mammary gland is a unique biosynthetic tissue that produces a variety of species-specific glycoconjugates, but the factors regulating the production of specific glycoconjugates are not well understood. To explore the underlying regulation, a fusion gene containing a cDNA encoding the human alpha 1,2-fucosyltransferase (alpha 1,2FT), which generates the H-blood group antigen, flanked by the murine whey acidic protein promoter and a polyadenylation signal, was introduced into mice. Milk samples from transgenic animals contained soluble forms of the alpha 1,2FT, as revealed by Western blots of milk samples using an anti-alpha 1,2FT antiserum and by the demonstration of alpha 1,2FT enzyme activity. Milk from transgenic animals also contained large quantities of 2'-fucosyllactose (Fuc alpha 1-2Gal beta 1-4Glc) and modified glycoproteins containing the H-antigen, whereas milk from control animals lacked these glycoconjugates. Expression levels of 2'-fucosyllactose were high in most animals and represented 1/3 to nearly 1/2 of the total milk oligosaccharides. These results demonstrate that heterologous transgenic expression of a glycosyltransferase can result in the expression of both the transgene and its secondary gene products and that the structures of milk oligosaccharides can be remodeled depending on expression of the appropriate enzyme. Furthermore, these results suggest that the lactating mammary gland may be a unique biosynthetic reactor for the production of biologically active oligosaccharides and glycoconjugates.