Cloning and expression of the cDNA sequence encoding the lysosomal glycosidase di-N-acetylchitobiase.

Di-N-acetylchitobiase (chitobiase) is a lysosomal glycosidase involved in the degradation of asparagine-linked glycoproteins. Previous studies have revealed that chitobiase is unique among lysosomal glycosidases in that it may not be expressed universally in mammals. In this study we have isolated full-length cDNA clones for human placenta and rat liver chitobiase. The cDNAs from both species encode a glycosylated polypeptide of approximately 40 kDa that displays chitobiase activity when expressed in COS-1 cells. By using the rat cDNA sequence as a hybridization probe, genomic DNA from several species was analyzed for chitobiase gene sequences. The results from these experiments suggest bovine and dog, two species that are believed to be chitobiase-deficient, maintain the chitobiase gene as part of their genetic load. The first three exons of the bovine chitobiase gene were cloned and found to encode an open reading frame that is 77% identical to both human and rat chitobiase. Northern blotting and amplification of mRNA by the polymerase chain reaction indicate that the chitobiase gene in bovine is functional, however, the level of expression is low. The presence of residual amounts of chitobiase enzyme activity in bovine liver and brain was demonstrated. Congruency of the very low levels of chitobiase enzyme to a similarly low level of chitobiase gene expression in bovine indicates that chitobiase in this species has a minor role in hydrolyzing the reducing end GlcNAc of asparagine-linked glycoproteins within the lysosomes. This is in contrast to a species such as human that express substantial quantities of this glycosidase. Thus, the extreme range of chitobiase gene expression among species explains why either 1 or 2 GlcNAc residues remain intact at the reducing end of stored oligosaccharides when either chitobiase-expressing or chitobiase-deficient species, respectively, suffers from a lysosomal storage disease.     

Rat liver chitobiase: purification, properties, and role in the lysosomal degradation of Asn-linked glycoproteins

Chitobiase, the lysosomal glycosidase responsible for splitting the GlcNAc beta-D-(1-4)GlcNAc moiety in Asn-linked glycoproteins, was purified over 600-fold from frozen rat livers utilizing an assay with di-N-acetylchitobiose as the substrate. The final preparation showed a major polypeptide of Mr 43,000 (sodium dodecylsulfate-polyacrylamide gel electrophoresis) that was determined to be the chitobiase by an immunological method. The purified chitobiase also hydrolyzed tri- and tetrasaccharides of chitin, which like di-N-acetylchitobiose were not substrates if first reduced by NaBH4. The initial products formed during hydrolysis of the tetrasaccharide were trisaccharide and GlcNAc. These results imply that chitobiase is a "reducing-end exohexosaminidase" which cleaves single GlcNAc units only from the reducing end of oligosaccharides. Fucose, typically found linked to the reducing-end GlcNAc in complex oligosaccharide chains, was found to block this reaction. Additional substrates that were hydrolyzed included GlcNAc beta-D-(1-4)MurNAc, the repeating structure from bacterial cell wall peptidoglycan, and the Man beta-D-(1-4)GlcNAc reducing-end component of glycoproteins. Km and Vm for hydrolysis of these substrates were of similar magnitude as for di-N-acetylchitobiose (6.3 mM and 15 mumol/min/mg protein, respectively). Liver tissues from nin mammalian species were surveyed for the presence of chitobiase activity. The activity was found in rat, mouse, rabbit, and guinea pig liver (Stirling [(1974) FEBS Lett. 39, 171-175] previously observed the enzyme in human liver), but not in dog, sheep, pig, cat, and cow liver. The presence or absence of chitobiase so far observed was found to exactly correlate with the type of oligosaccharide fragments found to accumulate in animals containing genetic or inhibitor-induced lysosomal storage pathologies. The presence of the chitobiase corresponds to the occurrence of one GlcNAc unit at the reducing end of stored oligosaccharides, while the absence of this glycosidase yields fragments with an intact GlcNAc beta-D-(1-4)GlcNAc moiety. These results verify our previous proposal that lysosomal disassembly of glycoproteins to free amino acids and sugars is an ordered, bidirectional pathway in which chitobiase (when present) catalyzes the last step during digestion of the protein-oligosaccharide linkage region.     

Translocation of Vibrio harveyi N,N''-diacetylchitobiase to the outer membrane of Escherichia coli.

The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Lysosomal degradation of Asn-linked glycoproteins

Catabolism of Asn-linked glycoproteins to monosaccharides and amino acids occurs in lysosomes. Break-down must be complete to avoid lysosomal storage diseases that occur when fragments as small as dimers are left undigested. Recent results have clarified several aspects of Asn-linked glycoprotein catabolism in mammals. First, degradation of the oligosaccharide portion is accomplished by exo-glycosidases, which act only from the nonreducing end of chains to release sugar monomers as products. In contrast, proteolysis can proceed from both end and internal points along the polypeptide to eventually yield free amino acids. A second important feature of the glycoprotein disassembly pathway is that the hydrolytic steps can be grouped into two sets of ordered reactions: I) stepwise hydrolysis of the major portion of the oligosaccharide chains by a set of exoglycosidases, and II) ordered disassembly of the protein and the oligosaccharide-to-protein linkage region. Process II can vary at a single reaction step depending on the species in which degradation takes place. Thus, the last step of reaction sequence II can be either: 1) hydrolysis of the actual peptide-to-carbohydrate linkage, or 2) removal of the reducing-end GlcNAc from a previously freed oligosaccharide. The latter cleavage is catalyzed by the lysosomal glycosidase chitobiase. Chitobiase has been found only in humans and rats and not in other mammals (dogs, cats, goats, sheep, cats, or cattle). The hydrolytic mechanism of this enzyme is unique as it appears to be a reducing-end glycosidase and can be viewed as an accessory step in the human and rat digestive pathways. The species that lack this enzyme likely rely on exo-beta-D-glucosaminidase to cleave GlcNAc from both outer chain residues and the chitobiose moiety at the protein-to-carbohydrate linkage.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

A porcine gene, PBK, differentially expressed in the longissimus muscle from Meishan and Large White pig

An investigation of differences in gene expression in the longissimus muscle of Meishan and Large White pigs was undertaken, using the mRNA display technique. A fragment of one differentially expressed gene was isolated and sequenced, whereupon the complete cDNA sequence was then obtained by using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of the gene is not related to any known porcine gene. Sequence analysis revealed that the open reading frame of this gene encodes a protein with 322 amino acids, thus displaying high sequence identity with the PDZ binding kinase (PBK) of eleven other animal species - dog, horse, cattle, human, chimpanzee, crab-eating macaque, rhesus monkey, rat, mouse, gray short-tailed opossum and platypus, so it can be defined as the porcine PBK gene. This gene was finally assigned GeneID:100141310. Phylogenetic tree analysis revealed that the swine PBK gene has a closer genetic relationship with the PBK gene of platypus. Gene expression analysis of eight tissues of a Meishan x Large White cross showed that the porcine PBK gene is differentially expressed in various tissues. Our experiment established the primary foundation for further research on this gene.     

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2

The bovine 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11β-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11β-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5′-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3′-untranslated region of 530 bp. Bovine 11β-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11β-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11β-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD⁺ and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11β-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.     

Identification of a bovine β-mannosidosis mutation and detection of two β-mannosidase pseudogenes

β-Mannosidase deficiency results in β-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine β-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3′ end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for β-mannosidosis carrier screening. Application of this test also revealed the presence of two β-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (>99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine β-mannosidosis phenotype suggests that the 22 C-terminal amino acids of β-mannosidase play an important role in the function of this enzyme. Received: 18 June 1999 / Accepted: 13 August 1999