Structure of the human gene for lysosomal di-N-acetylchitobiase

Bei Liu, Wasim Ahmad and Nathan N. Aronson, Jr.1

Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA

Received on August 20, 1998; revised on October 2, 1998; accepted on October 2, 1998

Chitobiase is a lysosomal glycosidase that acts during the ordered degradation of asparagine-linked glycoproteins to cleave the core chitobiose unit at its reducing end. Human chitobiase is expressed in significant amounts, while bovine chitobiase is produced at extremely low levels. To begin to understand this species-dependent expression, we determined the gene structure of human chitobiase. The human chitobiase gene (CTBS) is approximately 20 kb comprising seven exons varying from 0.1 to 2.3 kb and six introns of 0.3 to 8 kb. The previously characterized partial bovine chitobiase gene structure is similarly organized including exon and intron sizes and locations, but the human and bovine 5[prime]-flanking regions differ significantly. 5[prime]-RACE analysis of human chitobiase cDNA revealed only one transcriptional start site 45 bp upstream of the ATG translation initiation site. Computer analysis of the human chitobiase gene 5[prime]-flanking region shows characteristics of a typical housekeeping gene. The putative promoter region contains a distal TATA box, and there are several Sp-1 and AP-2 cis elements. In contrast, bovine chitobiase gene 5[prime]-flanking region shows totally different structures and may contain several silencers. A partial art-2 segment which is an artiodactyl Alu-like repetitive sequence, is also present. These evolutionary differences in the 5[prime]-flanking region of the chitobiase genes from human and bovine could account for the widely varied expression levels of the hydrolase within these two species.

Key words: di-N-acetylchitobiase/evolution/glycosidase/lysosomes

Introduction

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.

Results

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 1). Since no clone was found that contained both exon 6 and 7, another screening approach was used. Directional screening of the same genomic library by PCR using primer pairs from exons 5, 6, and 7 yielded an additional clone A3 harboring both exon 6 and 7 (Figure 1). Clones C1 and C4 contig contained exons 1 to 6; clone C3 contained exon 7 and 12 kb of its 3[prime] downstream region; and clone A3 contained exon 6 and exon 7 and the flanking regions (Figure 1). From these four overlapping genomic clones, the chitobase gene was deduced to consist of 7 exons ranging between 100 base pairs and 2.3 kb (Table I) spanning ~20 kb (Figure 1). The first exon contains 5[prime]UTR and 177 bp of coding region. The last exon (2.3 kb) is the longest and contains 198 bp coding sequence as well as the 3[prime]UTR. The 3[prime]UTR of the human chitobiase gene was not interrupted by an intron based on the identity of this genomic region to its cDNA 3[prime]-UTR. Intron lengths ranging from 0.3 to 8 kb separating the seven exons were determined by restriction endonuclease mapping and sizing gel analysis as well as by PCR and Long PCR analysis (Table I). The exon/intron boundaries of the human chitobiase gene were determined by sequencing from each exon into the flanking intron sequences. All splice-junction boundaries conform to consensus donor and acceptor sequences (Shapiro et al., 1987). Intron 6 is the longest intron and a previous consideration that another gene for a G-protein [gamma]5 subunit might be located within this intron (Fisher and Aronson, 1992b) was ruled out by PCR and sequencing (data not shown).


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. Sequences at intron-exon splice junctions of human chitobiase gene
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
Exon sequences are shown in uppercase letters and intron sequences in lowercase letters. The sizes of introns and exons are given in kilobases or base pairs.

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 2). The predicted transcriptional start site is a cytosine base -45 upstream of the ATG start codon. Inspection of the region upstream of this putative transcription start site revealed a TATA box and several highly GC-rich segments. The GC content was 84.2% for the region between -216 and the putative transcriptional start site. Analysis of the sequence by the program PROSCAN (Prestridge, 1995) predicted that a promoter is contained within the -216 to +34 region. Several Sp1 and AP-2 transcription factor binding sites are present in this region.


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 3). This predicted 91% complete bovine protein sequence has four potential Asn-linked glycosylation sites at positions 115, 193, 262 and 299, three of which are equivalent to those in human chitobiase. In comparison with the human and rat chitobiase amino acid sequences (Fisher and Aronson, 1992a) bovine chitobiase is less similar to human than is rat (Figure 3). The bovine amino acid sequence has an overall 71% identity to that of human chitobiase, and at the nucleotide level the identity of the coding regions of the two chitobiases is 81%. Sequence alignment resolves the amino acid difference into one major gap of five amino acids (human 275-279) missing from the bovine chitobiase. There are several regions that exhibit an even higher degree of conservation. One such conserved region is between amino acids 136-143 in all three proteins. The sequence comprises [beta]-strand four in the ([alpha]/[beta]8) barrel of the family 18 chitinases and includes the catalytic acid donor E143 (Davies and Hennrisat, 1995; Aronson et al., 1997). This glutamate is conserved in all glycosidase family 18 proteins that have enzymatic activity. Three other highly conserved sequences, 175-183, 202-210, and 246-259, also make [beta]-strands in the overall protein fold. Another enzyme segment of high identity in bovine (345-349) and in human (350-354) is [beta]8 of the barrel framework. This sequence was used to design a degenerate primer for successful PCR amplification of the low copy bovine cDNA (see Materials and methods). These [beta]-strands show significant homology among all known family 18 proteins (Aronson et al., 1997). A partial structure of the bovine chitobiase gene was reported earlier (Fisher and Aronson, 1992a) and genomic organization of the human chitobiase gene (Table I) is very similar. The seven exons and six introns of both genes have almost identical intron-exon border locations and the sizes of the corresponding introns from the two species, except intron 1, are similar. Bovine intron 1 is 1.2 kb while human intron 1 is 3.5 kb. The size of bovine intron 6 has not yet been determined.


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.

Discussion

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 1) and was previously localized to chromosome 1p22 (Ahmad et al., 1995). Both human and bovine genes consist of seven exons and six introns, and the 5[prime]UTR and 3[prime]UTR of human chitobiases are in exon 1 and exon 7, respectively (Table I, Figure 1). The highly GC-rich composition at the 5[prime]-flanking region of human chitobiase (Figure 2) suggests the housekeeping character of this gene. A high similarity of the coding region (Figures 3 and 4) but large difference in the 5[prime]-flanking region of human and bovine chitobiase genes (Figure 4) suggests that evolution has focused changes in the bovine promoter region (Figure 4) to cause low expression of the glycosidase. Specific trans-factors could be involved in this process, but cis-factors at the 5[prime]-flanking region likely play a crucial role in this process and could account for the near shut-down in bovine chitobiase expression (Aronson and Kuranda, 1989; Fisher and Aronson, 1992a).


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 4), share a high 77% identity in their coding region (Riise et al., 1997; Tollersrud et al., 1997), but the similarity of human and bovine [alpha]-d-mannosidase genes is a much higher 58% in the 5[prime]-flanking region upstream from the ATG start codon (Figure 4). No storage disease has ever been noted in humans or animals that would result from chitobiase deficiency, and it is likely that non-reducing-end hexosaminidases A and B can or do replace chitobiase in the digestion of the di-N-acetylchitobiose core. In this case, the genomic structure of chitobiase in artiodactyls and carnivores may provide scientists a rare view of a glycosidase gene undergoing evolutionary extinction.

Materials and methods

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.

Abbreviations

RACE, rapid amplification of cDNA ends; DONV, 5-diazo-4-oxo-norvaline; UTR, untranslated region.

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