Chitobiase (EC3.2.1.-) is a lysosomal glycosidase that hydrolyzes the reducing-end GlcNAc from the chitobiose core of oligosaccharides during the ordered degradation of asparagine-linked glycoproteins (Aronson and Kuranda, 1989; Aronson et al., 1989). This glycosidase can only act if the Asn that joins the oligosaccharide to protein is previously removed by glycosylasparaginase, making chitobiase the last step in lysosomal degradation of the protein/carbohydrate linkage component of Asn-linked glycoproteins (Aronson and Kuranda, 1989). In terms of evolution, chicken and slime mold have chitobiase activity, but about 80 million years ago during the late Cretaceous and Paleocene periods chitobiase activity in the process of mammalian species expansion decreased in ungulates and carnivora to a very low level, while primates, lagamorpha, and rodentia kept the enzyme (Aronson and Kuranda, 1989). This evolutionary break in chitobiase expression is evident from observed species-dependent variation in the structure of oligosaccharides left undigested in a number of lysosomal storage diseases. In human Tay-Sachs and Sandhoff's diseases, which result from lysosomal deficiency of non-reducing-end exo-hexosaminidases, only the penultimate N-acetylglucosamine residue remains at the reducing end of the stored oligosaccharides. However, in glycosidase-deficient species that lack chitobiase, such as bovine, oligosaccharides accumulate with their di-N-acetylchitobiose unit left intact (Abraham et al., 1983; Daniel et al., 1984; Barker et al., 1988). An important experimental observation to explain this species difference was the retention of GlcNAc-GlcNAc-Asn in a perfused rat liver treated with the compound DONV (Kuranda and Aronson, 1986). DONV specifically inactivates glycosylasparaginase and thereby indirectly blocks reducing end hydrolysis of GlcNAc by chitobiase. This experiment revealed that rat N-acetyl-[beta]-d-hexosaminidases must not efficiently hydrolyze the digestive intermediate GlcNAc-GlcNAc-Asn. Thus, in tissue lysosomes of species such as rat and human, chitobiase may be necessary to maintain an effective rate of complete degradation of the linkage region. In ungulates and carnivores it is possible that this hydrolase also plays an important biochemical role, but at a specific time or tissue location yet to be recognized in these chitobiase-deficient animals. We now report the structure of the human chitobiase gene and its comparison to that of bovine (Fisher and Aronson, 1992a). The DNA sequences in this article are available in GenBank with accession numbers AFO85711-714.
Genomic organization
By using a full-length 1.6 kb human chitobiase cDNA (Fisher and Aronson, 1992a) as a probe to screen a [lambda]-EMBL-3 genomic library, a [lambda]-contig (C1 and C4) of about 16 kb in length and a clone C3 containing exon 7 and the 3[prime] untranslated region were obtained (Figure
Figure 1. Cloning and structure of the human chitobiase gene. Top: the seven exons (1-7), six introns and 5[prime]-flanking region of the chitobiase gene are shown to scale. The solid bars represent coding exons and the open bars indicate 5[prime] and 3[prime] untranslated regions. Exon and intron sizes are in Table I. Each intron size except that of intron 6 was determined by PCR using converging primers from adjacent exons. The size of intron 6 was determined by both Southern hybridization and Long PCR. Bottom: partial restriction map (Bgl II and Nde I) of the chitobiase gene, showing regions encompassed by the contigs of [lambda]-clones used to determine the gene structure.
Table I.
Donor
Acceptor
Intron size (kb)
Exon size (bp)
222
TCGAG gt cagcg ..
.. ctggc ag GTCTT
3.5
139
TAAAG gt atgtt ..
.. tccac ag GAGAT
0.5
209
CACAG gt aaaaa ..
.. tccac ag GTAAC
3.5
172
AACTG gt aagac ..
.. tgact ag GATAT
1.0
100
GAGGA gt aggaa ..
.. tttct ag TCATG
0.3
160
ATAAA gt aagac ..
.. tatat ag GATCC
8.0
2.3
Alternative poly A usage
There are at least three mRNA species of 1.3, 1.6, and 3.2 kb for human chitobiase shown by Northern blot (Fisher and Aronson, 1992a). The 3[prime]UTR of the human chitobiase cDNA shows three AATAAA polyadenylation sites whose locations correspond with the length of these three messages. The results of RT-PCR also confirmed the existence of these three transcripts (data not shown).
Characterization of the 5[prime]-flanking region
To isolate the 5[prime] ends of the human chitobiase gene we performed 5[prime]-RACE. Due to a high GC content, a strong secondary structure might be expected to form at the 5[prime] end. Therefore, Tfl DNA polymerase which is engineered to read through GC-rich regions was used instead of Taq polymerase for PCR amplification. A 450 nt single band was obtained that suggests there is only one transcriptional start site. The PCR product was subcloned and sequenced (Figure
Figure 2. 5[prime]-Flanking region of human chitobiase gene. Closed circle shows the putative transcriptional start point determined by 5[prime]-RACE. 5[prime]-Distal to this transcriptional start point is a TATA box at -274 (underlined). A CACCC (-179) binding region and several CCCGCCC (-134, -121, -112, -107, -83, -52) sequences which are predicted to be Sp1 binding sites are also underlined. All of these elements are 5[prime]-upstream of the transcriptional initiation site at C(-45). The translational start ATG is in boldface type. Comparison of human and bovine chitobiase genes
To understand the poor expression level of chitobiase in species thought to have evolved after branching from a common lineage, we cloned bovine chitobiase cDNA by degenerate PCR. The resulting deduced partial bovine chitobiase protein sequence accounts for 351 of an expected 385 amino acids (Fisher and Aronson, 1992a) starting with the initiation methionine (Figure
Figure 3. Comparison of amino acid sequences of human, rat and bovine chitobiases. The alignment was done using the Wisconsin GCG 'Pileup" program. Consensus amino acids are shown in capital letters and the varied residues among the three species are presented in small letters above the dashes. Highly conserved regions among the species are highlighted. The vertical arrow indicates the putative signal peptidase cleavage site. N-Glycosylation sites in all three species are indicated by a boldface, underlined asparagine.
The present work describes the organization of the human chitobiase gene that is compared to its bovine counterpart (Fisher and Aronson, 1992a). The human gene spans 20 kb (Figure
Figure 4. Dot matrix comparison of human and bovine genomic regions (A) chitobiase (Fisher and Aronson, 1992a) and (B) [alpha]-d-mannosidase (Riise et al., 1997; Tollersrud et al., 1997). The analyses were performed as described by Maizel and Lenk (1981) using a window of 21 and stringency of 14. Coding sequences are shown in the upper-right quadrants, and 5[prime]-flanking sequences are in the lower-left quadrants.
We now note that the previously described 5[prime]-flanking/promoter region of the bovine chitobiase gene (Fisher and Aronson, 1992a) contains a partial artiodactyl Alu- type DNA segment. The bovine-specific region from -274 to -210 (AGTCGCTCAGTCGTGTCCGACTCTTTGCGACCCCATGGACTGCAGCACGCCAGGCCTCTCTGTCC) has high homology with 70 nt at the 5[prime]-terminus of the art-2 sequence (Duncan, 1987). It also has homology (in reverse direction) with the 50-nucleotide 3[prime]-terminus of a 120 nt repetitive unit present in the 5[prime]-flanking region and intron 1 of the bovine corticotropin-[beta]-lipotropin precursor gene (Watanabe et al., 1982). There are 100,000 copies of this 120 nt sequence in the bovine genome, and insertion of this Alu-like sequence in the promoter region of the chitobiase gene could have a role in repression of the bovine gene. On the other hand, from an evolutionary point this effect might have had an unknown beneficial impact on development of Artiodactyla and Carnivora, and virtual shutdown of chitobiase expression remained. For comparison, it is worth noting the sequence homology distribution of human and bovine genes for another lysosomal glycosidase. Lysosomal [alpha]-d-mannosidase is also involved in Asn-linked glycoprotein degradation, but it is equally well expressed in tissues of both these two species. The two mannosidase genes, like those for chitobiase (Figure Southern blot analysis of [lambda]-phage
Approximately 1 µg [lambda]-phage DNA was digested by an appropriate restriction enzyme and resolved on an 0.8% agarose gel. DNA was electroblotted from the gel onto a nylon membrane, and the resulting blot was hybridized with a gene-specific probe that had been random-primed labeled with biotinyl-11-dATP. Hybridization with 2 ng/ml of labeled probe was performed at 42°C in 4× SSC, 5× Denhardt's, 0.1% SDS, 50 µg/ml sheared herring sperm DNA. Posthybridization washes were conducted in the order: 2× SSC, 0.2% SDS for 15 min at room temperature, four times; and 0.1× SSC, 0.5% SDS for 10 min at 50°C. Genomic library screening by PCR or hybridization
A human [lambda]-EMBL-3 library was plated on 60 mm plates and grown for 8 h at 37°C at a density of 4 × 104 pfu/plate for PCR or 105 pfu/150 mm plate for hybridization. SM buffer was added to each plate to elute the phage, and PCR was done on each plate eluate. The positive phage pool(s) was further divided into 106 fractions with the lower complexity of 2 × 103 pfu per fraction. PCR screening was done on these clones and positive clone pools were picked for further screening by hybridization. Hybridization plates were cooled at 4°C for at least 1 h and overlaid with a nylon filter for 2 min, and the phage DNA was denatured. Membranes were auto-crosslinked and prehybridized in 4 × SSC, 1.0% SDS, and 50% formamide at 42°C for 2-24 h, and hybridized with 2 ng/ml labeled probe at 42°C overnight in 4× SSC, 4× Denhardt's, 0.1% SDS, 0.2 mg/ml sheared herring sperm DNA. Posthybridization washes were performed in the order: 2× SSC, 0.1% SDS, four times for 15 min at room temperature; 0.2× SCC, 0.1% SDS, one to four times at 42-65°C. DNA sequencing
Nucleotide sequencing was done by the dideoxynucleotide chain termination method using Circum-Vent DNA polymerase (New England Biolabs) according to the instructions of the manufacturer. Primers were end-labeled with [gamma]-[33P]ATP or [gamma]-[32P]ATP and each sequencing reaction was performed on a MJ Research thermal cycler programmed for the first reaction cycle for 3 min at 95°C, 1 min at 60°C, and 1 min at 72°C. The products were separated on a 5% polyacrylamide, 7.5 M urea gel. Long PCR
TaqPlus DNA polymerase (Strategene) was used to PCR amplify long introns. A 25 µl reaction mix contained the appropriate template (YAC clone Y1171 DNA, Ahmad et al., 1995; or [lambda] clone A3 DNA), reaction buffer, 30 mM dNTP, 20 pmol primers, and 5 U TaqPlus DNA polymerase. The reaction was carried out at 94°C for 2.5 min, followed by 30 cycles at 94°C for 45 sec, 60°C for 60 sec, and 71°C for 8 min, followed by a 30 min extension at 72°C. The two primer pairs used in this reaction were: NA 103(sense): 5[prime]-CGTCAGGTGCCCTACAAAACGATCATGAAG-3[prime] and NA 104(antisense): 5[prime]-GGGTTGGGGGAAAAGTATAGCACAAACTGC-3[prime]. 5[prime] RACE
Transcription initiation sites were determined by rapid amplification of cDNA ends (RACE) utilizing a Marathon cDNA RACE kit (Clontech) according to instructions of the manufacturer using the gene-specific primer NA151: 5[prime]-ACATTGATCCTGCTTTCAGAGCATCCTGGAT-3[prime]. Tfl DNA polymerase (Epicentre Technologies) was used to eliminate effects of possible secondary structure in this region of the DNA. For the primary PCR reaction, adaptor primer-1 (AP1) provided in the kit was used as the reverse primer. For the secondary PCR reaction, adaptor primer-2 (AP-2, nested to AP-1) provided in the kit was used as the reverse primer. The secondary PCR product was subcloned into pT7Blue-3 vector (Novagen) and sequenced. Cloning a partial cDNA for bovine chitobiase
In an attempt to clone the entire coding region of bovine chitobiase, RT-PCR was used to amplify the 3[prime] region of the gene. The template was a cDNA transcribed from bovine brain mRNA (Clontech) with Superscript II (Life Technologies). Three forward primers were used to clone and identify the PCR products: NA107: 5[prime]-GCATCCTGGATAGCTCAACAAG-3[prime]; NA108: 5[prime]-CATGCAAAAGGAGCCAGAGTAG-3[prime]; NA142: 5[prime]-ATGACTGGTCACAGATTACAAC-3[prime]. A degenerate antisense primer was designed according to the consensus sequence GIGMW of chitobiase which makes the [beta]8 strand in the common ([beta]/[alpha])8 barrel structure of family 18 chitinases (Aronson et al., 1997): NA145: 5[prime]-CARTTTGCRTTC-CACATNCC-3[prime] (R = G or A; N = G, A, T, or C). The first round of PCR was done using Taq DNA polymerase (Promega). A 25 µl reaction mixture contained the appropriate amount of template, reaction buffer, 5 mmol dNTP, 10 pmol primers (NA142 and NA145), and 1.25 U Taq DNA polymerase. The reaction was carried out at 94°C for 1 min and 30 sec, followed by 40 cycles at 94°C for 30 sec, 50°C for 50 sec, and 72°C for 1 min, with a final 30 min extension at 72°C. The PCR products were separated on a 2% agarose gel and the band of correct size was cut and soaked into 10 µl TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA). A 2 µl aliquot of this elute was used as template in the second round of PCR reaction using the forward nested primer NA107 or NA108 and the degenerate primer NA145. The PCR reaction conditions were the same as before except that the cycle number was decreased to 30. The final PCR product was subcloned into pT7Blue-3 vector (Novagen) and fully sequenced.
RACE, rapid amplification of cDNA ends; DONV, 5-diazo-4-oxo-norvaline; UTR, untranslated region.
Discussion
Materials and methods
Abbreviations
References
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