Department of Chemistry and Biomedical Sciences, University of Kalmar, SE- 391 82 Kalmar, Sweden
Received on June 16, 2004; revised on October 9, 2004; accepted on October 11, 2004
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Abstract |
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Key words: enzyme activity / expression / ß-hexosaminidase / lacrimal gland / sequencing
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Introduction |
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ß-Hexosaminidases are responsible for the degradation in acidic lysosomal compartments of glycoconjugated substrates derived from plasma membrane endocytosis. Because each subunit exhibits a different substrate specificity and catalytic activity, HexA is the only isoenzyme able to hydrolyze all known ß-hexosaminidase substratesGM2 ganglioside, ß-N-acetylglucosaminides, ß-N-acetylgalactosaminides, as well as 6-sulfated ß-N-acetylglucosaminides. The enzymatic degradation of GM2 ganglioside by HexA requires the assistance of the GM2 activator protein functioning as a cofactor in the reaction (Meier et al., 1991). The B isozyme has no catalytic activity toward GM2 gangliosides and sulfated compounds (Kytzia and Sandhoff, 1985
). The physiological significance of HexS has been questioned because only small amounts have been detected in human tissue, but recent studies have demonstrated that HexS is active on a wide range of compounds also hydrolyzed by HexA (Hepbildikler et al., 2002
).
The increased knowledge about ß-hexosaminidase is to a great extent due to studies of the - (HEXA) and ß-subunit (HEXB) genes in recent years. The nucleotide and deduced amino acid sequences that are of similar length in both subunits have been determined for human as well as mouse (Bapat et al., 1988
; Beccari et al., 1992
; Myerowitz et al., 1985
; O'Dowd et al., 1988
). The establishment of HEXA and HEXB gene organization also enabled scientists to learn more about the catalytic activity. Recent presentations of the crystal structure of HexB and of the GM2 activator protein have provided additional information about the hydrolytic mechanism for all isoenzymes (Mark et al., 2003
; Wright et al., 2000
). Of even greater importance were perhaps the identification of the genetic defects causing the inherited lysosomal storage disorders Tay-Sachs (Myerowitz and Hogikyan, 1986
) and Sandhoff diseases (O'Dowd et al., 1986
). Tay-Sachs is caused by mutations in the HEXA gene on chromosome 15; Sandhoff's disease is due to mutations in the gene coding for HEXB. Defects in either gene, results in accumulation of the normal substrate for HexA, GM2 ganglioside, in cells. The compound is particularly abundant in nerve tissue, resulting in a progressive neurodegeneration. Rarely, GM2 gangliosidosis can also be caused by mutations in the GM2 activator protein gene, called the AB variant form (Mahuran, 1999
).
Studies of ß-hexosaminidase in the lacrimal gland were initially performed in belief that the enzyme would function as a lysosomal marker in membrane trafficking experiments. It was reported that ß-hexosaminidase activity showed a higher steady-state content in endoplasmic reticulum and the Golgi compartments, compared with the lysosomal enzyme cathepsin B (Gierow et al., 1996; Hamm-Alvarez et al., 1997
). ß-Hexosaminidase also shows the highest catalytic activity compared with the hydrolases, ß-glucuronidase, aryl sulfatase, and
- and ß-galactosidase as well as
-fucosidase measured in lacrimal gland acinar cell fluid (Gierow et al., 2001
; Sjögren et al., 2000
), demonstrating the importance of exploring the role of ß-hexosaminidase in tear fluid. Today, the catalytic activity of ß-hexosaminidase commonly serves as a marker of regulated secretion in studies of rabbit lacrimal gland acinar cells in primary culture (Gierow and Mircheff, 1998
; Hamm-Alvarez et al., 1997
; Yang et al., 1999
). Secretion from lacrimal acinar cells is regulated through several signaling pathways, including both IP3/DAG and cAMP formation (Hodges and Dartt, 2003
). Stimulation of both pathways simultaneously triggers a maximal secretory respons from cultured acinar cells (Gierow et al., 1995
). ß-Hexosaminidase release parallels total protein secreted by stimulation of these pathways (Gierow et al., 1997
).
The purpose of the present study was to further characterize the enzyme ß-hexosaminidase in rabbit lacrimal gland. Currently, there are no antibodies available that recognizes rabbit ß-hexosaminidases, which would have been a useful tool in intracellular trafficking studies. To elucidate any variations at the DNAprotein level, in different species, the nucleotide sequences for rabbit ß-hexosaminidase - and ß-subunits were determined, showing high identity with human and mouse sequences. Northern blot analysis demonstrates an up-regulated
-subunit expression in cultured acinar cells compared with lacrimal gland tissue, which is not detected for the ß-subunit. Enzymatic studies showed a significantly higher enzymatic ß-hexosaminidase activity in cellular extracts, and determination of the HexA and HexB proportions revealed that HexA is the major active ß-hexosaminidase isoform in cells and lacrimal gland tissue.
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Results |
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Discussion |
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The lacrimal gland is believed to be the main source of these enzymes, demonstrated by a catalytic activity that can be correlated to that determined in tear fluid, at a low pH (Van Haeringen and Glasius, 1976, 1980
). Despite the relatively high activity of ß-hexosaminidase measured in tear fluid, the question about the functional role for this enzyme at the ocular surface, which has a neutral pH, remains unanswered. Not considering the acidic requirements for activity against the artificial substrates, ß-hexosaminidase could participate in the turnover of mucins in the tearfilm. Also, the pH of the mucin microenvironment is not known. Abnormalities in the mucus layer, as a result of longer oligosaccharide chains on glycoproteins, have been observed in a canine model of keratoconjunctivitis sicca (Hicks et al., 1998
). Preliminary results indicate that purified ocular mucins from rabbit are degraded by enzymes secreted by cultured lacrimal cells (Matthews et al., 2001
). Cholinergic stimulation with carbachol accelerates both release of secretory vesicles and the membrane recycling by endocytosis in acinar cells (Gierow et al., 1995
; Lambert et al., 1993
). After stimulated exocytosis a disappearance of ß-hexosaminidase from secretory membrane compartments has been reported at the same time as an increasing amount could be detected in trans Golgi compartments (Yang et al., 1999
), suggesting that a large amount of ß-hexosaminidase is recycled back (Hamm-Alvarez et al., 1997
). The mechanism by which a significant portion of lysosomal hydrolases are discharged extracellularly is still not known but it could be a process by which enzymes are concentrated to lysosomal compartments trough reinternalization by mannose 6-phosphate receptors.
The possibility that ß-hexosaminidases, escaping from the lysosomal route, are secreted in their nonmature form and then recaptured for final processing in the intracellular acidic membrane compartments has been addressed in attempts to restore Tay-Sachs defects by overexpressing the -subunit in fibroblasts in vitro and then allowing for recapture in HexA deficient fibroblasts (Guidotti et al., 1998
; Martino et al., 2002
). This is also in agreement with the study showing that ß-hexosaminidase in human serum only exists in its precursor form (Isaksson and Hultberg, 1995
). The precursor form of HexA can degrade GM2 ganglioside in the presence of GM2 activator protein at the same rate as the mature HexA isoenzyme (Hasilik et al., 1982
), suggesting that the secretory, nonmature portion of ß-hexosaminidase is functionally active in the extracellular fluid. Furthermore, a large fraction of the GM2 activator protein is secreted (Rigat et al., 1997
). It has been shown to bind HexA at pH 7 (Yadao et al., 1997
) and function as a glycolipid transporter at physiological pH (Smiljanic-Georgijev et al., 1997
), suggesting that the GM2 activator protein could induce the hydrolysis of GM2 gangliosides through interaction with HexA even at neutral pH. Whether the catalytical activity of ß-hexosaminidase, observed in the secretory fluid from rabbit lacrimal gland acinar cells, arises from proteolytically processed enzymes or the nonmature form remains to be explored. Though only insignificant ß-hexosaminidase activity at pH 7.0 has been detected in human tear fluid and serum (Van Haeringen and Glasius, 1976
) as well as fluid secreted by rabbit lacrimal gland acinar cells (data not shown).
Northern blot studies of ß-hexosaminidase -subunit clearly show a higher mRNA expression in cultured lacrimal gland acinar cells compared with tissue. This could be the result of purification of acinar cells from other structures with small amounts of or no ß-hexosaminidase, or the culturing environment, affecting the gene expression within the acinar cells. Isolated cells were cultured in serum-free medium, a condition previously shown to up-regulate both
- and ß-subunit mRNA expression in microglial cells (Beccari et al., 1997
). An up-regulated
-subunit transcription and protein synthesis followed by an increased enzyme secretion could be an attempt for the cells to modulate the surrounding matrix and stabilization of acinar structures. An interesting feature is that the mRNA expression studies of the ß-subunit, present in the functionally active isoenzymes HexA and HexB, show an even expression between cultured cells and tissue. High levels of the
-subunit could be necessary to favor HexA formation instead of the more stable HexB isoform as discussed by Mahuran (1995)
. The fact that no difference could be observed at the mRNA level after stimulated secretion suggests that ß-hexosaminidase is stored in secretory vesicles at the apical membrane ready to be released. Studying the expression for a longer period of time (hours) after stimulation, resulting in emptying of intracellular ß-hexosaminidase stores, would perhaps reveal changes at the mRNA level.
Enzymatic assays revealed a slightly higher ß-hexosaminidase activity in cellular extracts, which together with the northern blot data indicate that the -subunit and consequently HexA is present to a higher degree than HexB in primary cultured acinar cells. Heat inactivation of HexA, resulting in an
93% loss of both total ß-hexosaminidase and HexA activity, suggests that HexA is the major active ß-hexosaminidase isoform in the lacrimal gland.
Sequence analyses were performed to explore any differences between rabbit and the species human and mouse, at the DNAprotein level. Comparison of the translated peptide sequences with mouse and human clearly showed that the overall identity, glycosylation sites, and proposed catalytic sites are highly conserved both for - and ß-subunits between all three species. Absence of the human site of glycosylation at Asn142 in the rabbit ß-subunit could be the reason why antibodies directed against the human protein do not detect the rabbit ß-hexosaminidase. Despite several attempts, with different molecular biology approaches, we could not obtain the 5' ends of either subunit. Comparative analysis of the sequence data will though be useful in selecting rabbit specific subunit peptide epitopes, likely to be surface exposed on the mature enzyme, which will be used for production of antibodies.
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Materials and methods |
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Cell purification and culture
Lacrimal gland acinar cells were isolated from female New Zealand White rabbits weighing 1.72.0 kg (ESF-Products, Estuna AB, Norrtälje, Sweden) as described previously (Gierow et al., 1996). Animals were handled according to directions from the Ethical Committee for Animal Experiments (Linköping, Sweden) and the ARVO statement for use of animals in ophthalmic and vision research. Purified single cells were cultured on Matrigel (40 µg/ml) coated wells with a cell density of 6.5 x 105 cells/cm2 in PCM, a serum-free Ham's F-12 (Invitrogen) and low-glucose Dulbecco's modified Eagle's medium (Invitrogen) in a 1:1 mixture and supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, 0.1 mM sodium citrate, a mix of 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml sodium selenite, 2 mM sodium butyrate, 5 nM hydrocortisone, and 0.3 µM linoleic acid.
Cell and tissue treatment
Treatments were performed after 2 days in culture, allowing cells to reorganize into acinus-like structures with distinct apical and basolateral regions. To be able to study the ß-hexosaminidase - and ß-subunit expression both in resting cells and after maximal stimulated secretion, acinar cells were incubated in the presence or absence of 0.1 mM carbachol and 0.1 µM VIP in Hank's balanced salt solution (Sigma-Aldrich), supplemented with 10 mM HEPES and 1 mM CaCl2, with final pH 7.6, for 1 h at 37°C. Following stimulation, supernatants were collected from the culturing dishes, and detached cells were removed by brief centrifugation. Cells were lysed in Hank's balanced salt solution, supplemented as stated, containing 1% Triton-X 100; scraped; and saved at 80°C until use. To assay enzyme activity,
100 mg tissue was homogenized using an Ultra-Turax homogenizer (Janke & Kunkel, Staufen, Germany) in Hank's balanced salt solution, supplemented as stated, containing 1% Triton-X 100; filtered through a Nylon Net Filter, 180 mm (Millipore, Billerica, MA); and saved at 80°C until use.
Enzymatic assays
Secretion from resting and stimulated lacrimal gland acinar cells was measured using total ß-hexosaminidase activity as marker (Gierow and Mircheff, 1998). To evaluate the proportions of HexA and HexB activity in cell extracts and tissue samples, HexA was thermally inactivated by heating samples for 1 h at 52°C (Martino et al., 2002
). Protein content was measured according to a modified method of Lowry et al. (1951)
, described earlier (Gierow et al., 1995
). Total ß-hexosaminidase enzyme activity, referring to the hydrolysis by HexA and HexB isoenzymes in samples was determined using the substrate 4MUGlcNAc (7.5 mM) and HexA-specific activity was confirmed with 4MUGlcNAc6SO4 (0.1 mM) using the method by Barrett and Heath (1977)
. Routinely, reactions are started by adding 50 µl reaction buffer (133 mM sodium citrate, 133 mM sodium chloride and substrate, pH 4.3) to the samples. After 2 h the reaction is terminated by addition of 2 ml quench solution (50 mM glycine and 5 mM ethylenediamine tetra-acetic acid, pH 10.5). Absorbance measured at 460 nm using Flourolog 3-22 Florescence Spectrophotometer (Instruments S.A., Edison, NJ) was calibrated to a 4-methylumbelliferone standard (0.1 mM) concentration subjected to the same conditions. Data were analyzed by Student t-test, where p < 0.05 was considered statistically significant.
RNA isolation
Total RNA was extracted from cultured resting and stimulated acinar cells and 0.2 g rabbit lacrimal gland tissue using the Ultraspec II RNA kit (Biotecx Laboratories, Houston, TX), if not otherwise stated. The tissue sample had been snap-frozen in liquid nitrogen on removal and kept at 8°
C until further processing according to the manufacturers instructions.
Construction of complementary cDNA clones
Using 1 µg total RNA extracted from lacrimal gland tissue, single-stranded random hexamer-primed cDNA was generated with the GeneAmp RNA polymerase chain reaction (PCR) kit (Perkin Elmer, Roche Molecular Systems, Branchbury, NJ). The ß-hexosaminidase - and ß-subunit-specific cDNA fragments were PCR amplified with the following primer pairs; HexA Fw1/HexA Rw1 and HexB Fw1/HexB Rw2, respectively (See Table II for primer nucleotide sequence). Primers were designed from highly conserved regions within human (GenBank accession number NM000520) and mouse (GenBank accession number NM010421) sequences of ß-hexosaminidase
-subunit and human (GenBank accession number NM000521) and mouse (GenBank accession number MMHEXB) sequences of ß-hexosaminidase ß-subunit. The cDNA fragments, corresponding to the ß-hexosaminidase
- (394 nt) and ß- (343 nt) subunits, were separated on an ethidium bromidestained 1% agarose gel and purified with Jet Quick Purification Spin Kit (Genomed, Bad Oeyenhausen, Germany), cloned into pGEM-T vectors (Promega, Madison, WI), and finally transformed into competent Escherichia coli JM 109 cells (Promega). Plasmid DNA was purified with Wizard Plus mini- and midiprep kits (Promega) and sequenced using the ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, CA) and the BigDye Terminator Cycle sequencing ready reaction DNA Sequencing kit (PE Applied Biosystems, Warrington, U.K.).
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Northern blot analysis
RNA separation, transfer, and probe hybridization followed the procedure described in detail earlier (Gierow et al., 2002; Magnusson et al., 2001
). Briefly,
15 µg total RNA isolated from lacrimal gland tissue or cultured resting and stimulated acinar cells was separated on a denaturating formaldehyde-agarose gel by electrophoresis. Comparable amounts RNA loaded were visualized by UV illumination of ethidium bromideinduced fluorescence of 18S and 28S ribosomal RNA bands. Thereafter RNA was transferred onto an uncharged nylon transfer membrane (0.45 Micron, Micron Separations, Westboro, MA) and cross-linked by UV illumination. Blots were prehybridized for at least 2 h, followed by hybridization with 32P-labeled antisense RNA probes against the ß-hexosaminidase
- or ß-subunit overnight at 65°C. Blots were then rinsed, and hybridization signals were detected by autoradiography and exposures were made at 80°C using Kodak X-OMAT AR or Kodak Biomax MS film. Densitometric quantification of mRNA expression from northern blots was performed as described earlier (Magnusson et al., 2003
). Alterations in expression were calculated by comparing cultured cell data to lacrimal tissue data, set to 1.0. Data were analyzed by Student t-test, where p < 0.05 was considered statistically significant.
Sequencing of the ß-hexosaminidase -subunit
Determination of the sequence of the ß-hexosaminidase -subunit was performed with the 3'-RACE and the 5'-RACE systems according to the manufacturers protocol (Invitrogen). All PCR products were analyzed on ethidium bromidestained 1% agarose gels. In the 3'-RACE, first strand cDNA was synthesized from 2.9 µg total RNA using the supplied adapter primer (AP). PCR amplification was performed with the gene-specific HexA Fw1 primer and the supplied abridged universal amplification primer (AUAP). Nested PCR reactions were performed with the obtained PCR product as a template, using HexA Fw2 or HexA Fw4 primers in combination with the AUAP primer. Amplified fragments of expected size, 1500 bp and 500 bp were purified, cloned, and sequenced as described. In addition, sequencing was also performed directly on the purified 1500-bp 3'-RACE amplified cDNA with the forward primers HexA Fw2 and HexA Fw3. In the approach to amplify the 5' end of the rabbit
-subunit, total RNA was isolated from 0.3 g lacrimal gland tissue, using the GenElute Mammalian Total RNA Kit (Sigma-Aldrich). First strand cDNA was synthesized from 3.4 µg total RNA with Hex Rw1 as reverse primer. PCR amplification of dC-tailed cDNA was performed using the puReTaq Ready-To-Go PCR Beads kit (Amersham Biosciences, Piscataway, NJ) with the supplied forward abridged anchor primer and HexA Rw2 reverse primer. A nested PCR was performed using AUAP and the HexA Rw3 primer. An amplified cDNA fragment of 550 bp was purified, cloned, and sequenced as described.
Sequencing of the ß-hexosaminidase ß-subunit
To amplify the 3' end of the ß-hexosaminidase ß-subunit mRNA, the 3'-RACE System, Version C kit (Invitrogen) was used. First strand cDNA was synthesized from 11 µg total RNA with the supplied AP. Superscript III Reverse Transcriptase (Invitrogen) was used instead of the enclosed Superscript II Reverse Transcriptase. Gene-specific primers were designed from highly conserved regions within human (GenBank accession number NM000521) and mouse (GenBank accession number NM010422) sequences of ß-hexosaminidase ß-subunit. PCR amplification of synthesized cDNA was performed with the HexB Fw1 forward primer and the supplied AUAP, as reverse primer. The PCR product was analyzed by ethidium bromidestained 1.0% agarose gel electrophoresis, and a fragment of expected size was cut out and dissolved in 100 µl H2O at 94°C. A nested PCR was carried out, using the gel matrix fragment as a template and the AUAP primer and the HexB FwJ1 as forward primer. The amplified cDNA fragment of 1140 bp was purified, cloned, and sequenced as described. Because of the relative large size of the fragment, another forward primer, HexB Fw3, was designed to obtain sequence from the middle section of the fragment.
Sequence analysis
NCBI's BLAST program (www.ncbi.nlm.nih.gov/BLAST) for nucleotide sequences was used for confirming sequence similarities of obtained rabbit nucleotide sequences with published human and mouse ß-hexosaminidase - and ß-subunit nucleotide sequences. Nucleotide sequence alignment and analysis was performed with the Vector NTI Suite 7.0 program. The Clustal W 1.8 program (www.ebi.ac.uk/clustalw) was used for multiple alignments of amino acid sequences with a gap open penalty of 10.0 and a gap extension of 0.1.
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Acknowledgements |
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Abbreviations |
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References |
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