verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis

GASTROENTEROLOGY 1995;109:1234-1240

Verification of the Lactase Site of Rat Lactase-Phlorizin Hydrolase by Site-Directed Mutagenesis

ADRIANA M. NEELE,* ALEXANDRA W. C. EINERHAND,* JAN DEKKER,* HANS A. BULLER,* JEAN-NOEL FREUND, t MENNO VERHAVE, § RICHARD J. GRAND, § and ROBERT K. MONTGOMERY § §Division of Pediatric Gastroenterology and Nutrition, Floating Hospital for Children, and the Center for Gastroenterology Research on Absorptive and Secretory Processes, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts; tlNSERM Unit6 61, Strasbourg, France; and *Division of Pediatric Gastroenterology and Nutrition, Academic Medical Center, Amsterdam, The Netherlands

Background & Aims: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. Methods: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. Results: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. Con- clusions: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

L actose is the major carbohydrate in most mammalian milk. After ingestion, lactose is hydrolyzed to glu-

cose and galactose by lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23 and 3.2.1.62), located on the apical membrane of small intestinal absorptive epithelial ceils. LPH has long been known to have glycosylceramidase activity, 1-3 as well as to display both ~-galactosidase and ~- glucosidase activities against a number of substrates. 4 In addition to these earlier data obtained using biochemi- cally purified enzymes, our laboratory showed that the single LPH protein isolated with a monoclonal antibody hydrolyzed multiple substrates) These findings have led to numerous attempts to identify the active site(s) on the protein and to delineate mechanisms of hydrolysis.

Data from a number of laboratories have indicated that LPH has two separate active sites. 4'6'7 The two sites are distinguished by differential heat inactivation, different inhibitor sensitivity, and incomplete mutual substrate inhibi- tion. These analyses suggest that both sites hydrolyze ~- glucopyranosides and ~-galactopyranosides, the lactase site preferring hydrophilic aglycones such as lactose and cellobiose and the phlorizin hydrolase site preferring hydrophobic ones such as phlorizin, glycosylceramides, 0-nitrophenyl-~- D-glucopyranoside, and other aryl-~-glycosides. 3'8 Recently, it has been shown that the active site-directed inhibitor conduritol ~-epoxide (CBE) binds to rabbit LPH in a 2:1 molar ratio, consistent with the presence of two active sites on each LPH molecule. 8

Wi th the cloning and complete sequencing of human, rabbit, and rat LPH, it is now established that LPH consists of four domains that are highly homologous at both the DNA and amino acid levels. 9'1° The precursor form is processed by the enterocyte to remove N-terminal domains I and II; the mature enzyme consists of only C- terminal domains III and IV. From the sequence homology of LPH to bacterial glucosidases with known active sites, Henrissat 11 predicted that glutamic acids at amino acid position 1273 and 1749 in human LPH would be the nucleophiles of the domain Ill and IV active sites. This model was supported experimentally by Wacker et al.,8 who showed binding of [3H]CBE to the corresponding residues (1271 and 1747) in rabbit LPH. These investigators showed that CBE binding inactivated both the lactase and phlorizin hydrolase activities of LPH. The identity of the binding sites was confirmed by direct sequencing of peptide fragments that had the labeled CBE bound to glutamic acid residues 1271 and 1747.

Abbreviations used in this paper: CBE, conduritol I~-epoxide; DMEM, Dulbecco's modified Eagle medium; LPH, lactase-phlorizin hydrolase; ONP-gal, o-nitrophenyl-~-l,4-galactopyranoside; ONP-glc, o-nitrophenyl-~l,4-glucopyranoside; SDS, sodium dodecyl sulfate,

© 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

October 1995 RAT LACTASE-PHLORIZIN HYDROLASE ACTIVE SITES 1235

Based on CBE-binding kinetics and inactivation profiles,

these investigators identified E1271 as the lactase site and E1747 as the phlorizin hydrolase site.

The correlations of enzyme activity and active site are

all derived from indirect analyses, and some of the avail-

able enzyme kinetic data are difficult to explain on the

basis of one lactase and one phlorizin hydrolase site.

Therefore, we under took to test the hypothesis directly,

using site-directed mutagenesis to change the glutamic

acid residues of the putative active sites in rat LPH to

aspartic acid or glycine. W e predicted that, if the model

were correct, an E 1 2 7 4 G mutan t would eliminate lactase

activity only, whereas an E1750G mutan t would elimi-

nate phlorizin hydrolase activity only. However, these

predictions were not borne out. Instead, in the present

study, we report data consistent with the hypothesis that

LPH has a single catalytic site for both hydrophil ic and

hydrophobic aglycones located in domain IV, represent-

ing the lactase and the majori ty of the phlorizin hydrolase

activities, and a second domain III site for hydrophobic

aglycones only, showing no lactase but low phlorizin

hydrolase activity.

Mater ia ls and Me thods

Construction of Full-Length Rat LPH Complementary DNA in Expression Vector pcDNAI

The nearly full-length LPH complementary DNA (cDNA) used, pRLU61, was described previously. 9 The polymerase chain reaction was used to construct a full-length cDNA from it and to remove the poly-A sequence. A genomic clone containing intron 1 and exon 1 of rat LPH was used as the template to generate 29 nucleotides missing at the 5' end, whereas pRLU61 was used as the template for the 3' end. The polymerase chain reactions generated two cDNA fragments: one for the 3' end that was digested with Xba I and NotI, resulting in a fragment containing nucleotides 4376-6125, and one for the 5' end that was digested with EcoRI and HindlII, resulting in bases 1-585. Fragment 4376-6125 Xba I through NotI was inserted into the corresponding unique sites of the pRLU61 plasmid. Subsequently, the rat LPH cDNA, still lacking the 5' end, was isolated after digestion with EcoRI and NotI and cloned into a pcDNAI expression vector (Invitrogen, San Diego, CA) that had the unique HindlII site inactivated. This was followed by the ligation of the 1 - 585 EcoRI through HindlII fragment into the rat LPH cDNA, resulting in a full-length rat LPH cDNA in the pcDNAI expression vector. Validity of the constructs was confirmed by sequencing with the dideoxy chain termination method 12'*3 using a Sequenase kit (United States Biochemical, Cleveland, OH) according to the manufacturer's instructions.

Site-Directed Mutagenesis

Mutants are identified by indicating the normal amino acid by the single letter code, numerical position in the amino

acid sequence, and then the mutated amino acid. To test the identity of the active sites, we chose to make two mutations of the previously identified glutamic acids. A minimal change was made by converting the glutamic acid to aspartic acid, which has the identical structure except for a side chain shorter by one carbon. The major change was conversion of glutamic acid to glycine, which completely eliminates the side chain believed to be involved in substrate interaction, replacing it with a hydrogen. We predicted that if the identified glutamic acids were the critical nucleophiles of the active sites, one or both of these mutations should eliminate the activity of the mutated site. Two additional mutants, Y1271F and T1273P, were examined to determine the effect of changes in amino acids close to the nucleophile on lactase activity and protein antigenicity. Site-directed mutagenesis was performed as described by Kunkel I~ and Kunkel et al. .5 using VCSM13 phage and the following mutant sense primers (the nude otides changed are underlined): E1274G, 5'-CCTATTTATATC- ACTG__GAAATGGACAGGGAC- 3 ' ; E 1750G, 5 ' -CGATTT- ATGTCACCGGGAATGGTGTCTCCA-3 ' ; E 1274D, 5 '- C T A T T T A T A T C A C T G A C A A T G G A C A G G G A C T - 3 ' ; E 1750D, 5 ' -CGATTTATGTCACCGACAATGGTGTCT- CCA-3'; Y1271 F; 5 ' -GCAACATTCCTATTTTTATCACTG- AAAATGG- 3 '; and T 1273 P, 5'CATTCCTATTTATATCCC- TGAAAATGGACAG-3' . The mutations were confirmed by DNA sequencing using the following sense primers: SEQ.MUT.1, 5 ' -TGTGATGCACCAAGACG-3' ; and SEQ.- MUT.3, 5 ' -TTGCAGACAGCTCTTGG-3' .

Transient Transfection of COS-7 Cells

The COS-7 ceils were obtained from the American Type Culture Collection. COS-7 ceils were cultured in Dulbecco's modified Eagle medium (DMEM; GIBCO/BRL, Gaithersburg, MD) containing 5 % fetal calf serum. Twenty-four hours before transfection, 1.5 × 106 COS-7 cells were seeded in 100-mm dishes. The cells were rinsed once with DMEM without fetal calf serum. Subsequently, 2 mL DMEM containing 0.125 mmol/ L Tris/HC1, pH 7.2; 10% Nu-serum (Collaborative Research, Bedford, MA); and 5 gg wild-type or mutated rat LPH cDNA in the pcDNA expression vector was added to the ceils. A mock transfection (no DNA) was also performed as a control. After 1 minute, DMEM containing 0.125 mmol/L Tris/HCl, pH 7.2; 10% Nu-serum; and 3.56 mmol/L diethylaminoethyl-dextran was added. This was incubated for 4 hours at 37°C in 5% CO2. Thereafter, the dextran media were aspirated, and the cells were rinsed with phosphate-buffered saline containing 10% dimeth- ylsulfoxide. Then 6 mL of DMEM with 5% fetal calf serum and 0.1 mmol/L chloroquine (Sigma Chemical Co., St. Louis, MO) was added and again incubated for 4 hours at 37°C. The cells were then rinsed once with DMEM containing 5% fetal calf serum and incubated overnight at 37°C in 8 mL fresh DMEM with 5% fetal calf serum. After 36 hours, the ceils were harvested by scraping and immediately put on ice. The cells were solubilized in 20 mmol/L HEPES, pH 7.5; 20 mmol/L NaC1; 0.02% NAN3; 0.5% Triton X-100; leupeptin (10 btg/ mL); soybean trypsin inhibitor (100 [.tg/mL); phenylmethylsul- fonyl fluoride (1 mmol/L); and pepstatin A (10 btg/mL).

1236 NEELE ET AL. GASTROENTEROLOGY Vol. 109, No. 4

A

=i, ,, =¢., ,,~

205 k D - -

116.5 k D - -

80 k D m

49.5 k D

L P H

I g G

Figure 1, SDS-PAGE analysis of immunoprecipitated wild-type and mutant LPH. LPH proteins were immunoprecipitated as described in Materials and Methods. Samples were electrophoresed on a 4% stacking and a 7.5% polyacrylamide separating gel. The gels were stained with Coommas- sie blue. (A) Immunoprecipitates used for analysis are shown. Lane 1, prestained high-molecular-weight markers 205, 116.5, 80, and 49.5 kilodaltons (kDs); lane 2, control cells with mock transfection with no DNA; lane 3, wild-type LPH; lane 4, E1274G single mutant LPH; lane 5, E1750G single mutant LPH; and lane 6, double mutant LPH containing both E1274G and E1750G. Every lane, except the control, shows the high-molecular-weight LPH precursor (220 kilodaltons) and the immunoglobulin G (IgG) heavy chain (55 kilodaltons). (B) Reimmunoprecipitation as described from the supernatants. Lanes are the same as in A.

Preparation of Microvillus Membranes

Microvillus membranes were isolated from the rat small intestine by the Cai+-precipitation method. 16

Immunoprecipitation

The monoclonal antibody against rat lactase was pre- pared and used as described previously. 17 A standard amount of antibody coupled to Sepharose CL-4B beads (Pharmacia, Brussels, Belgium) was used to immunoprecipitate LPH from rat microvillus membranes and from transfected COS-7 ceils. LPH was immunoprecipitated overnight at 4°C. The LPH bound to the Sepharose beads was recovered by centrifugation and washed thoroughly. To show that the amount of antibody was l imiting and that the assays were performed with equal amounts of LPH, the supernatant from this immunoprecipitation was immunoprecipitated again. The beads were washed with 50 mmol/L Tris/HC1, pH 7.4; 150 mmol/L NaC1; 0.05% Triton X-100; and 0.1% sodium dodecyl sulfate (SDS) and 50 mmol/L Tris/HC1 pH 7.4; 150 mmol/L NaC1; 0.1% Triton X-100; and 0.02% SDS. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) I8 or used for enzyme assay. We confirmed our previous demonstration 5 that rat LPH bound to these antibody beads has un- changed enzyme activity. The measured units of enzyme activity bound to the antibody beads after precipitation were equal to the units removed from solution by the immunoprecipitation.

Enzyme Activity Assays

Lactase activity was measured according to Dahlqvist 19 using ~-D-glucopyranosyl(1-->4) ~-D-glucopyranose (cellobi-

ose) and ~-D-galactopyranosyl(1-->4) ~-D-glucopyranose (lactose) (Fluka, Buchs, Germany) as substrates. The tactase activity of rat LPH may be as much as 50-fold higher than phlorizin hydrolase activity. 4 Because LPH hydrolyzes phlorizin at a very

low rate, numerous natural and artificial substrates, for which LPH has varying affinities and hydrolytic activities, have been used to measure phlorizin hydrolase activity. 1'4'3'7 Because they

showed relatively high levels of activity, we chose to measure phlorizin hydrolase activity using two synthetic substrates: 0- nitrophenyl-~-l,4-galactopyranoside (ONP-gal) and 0-nitrophenyl-~-l,4-glucopyranoside (ONP-glc) (Sigma Chemical Co.) as described previously. 4 Because activities were higher with ONP-glc, these data are presented. Identical patterns were observed with ONP-gal (data not shown). For each assay, one plate of transfected COS-7 ceils was used. Alt assays are timed incubations in the presence of excess substrate, except for the Michaelis constant (K~) determinations. Activities shown are averages of 3 - 6 individual assays. Activities of each mutant were expressed relative to that from cells transfected with wild-type LPH in the same experiment. Because equal amounts of immunoprecipitated LPH were assayed, it is unlikely that the measured differences in activity reflect differences in processing or turnover of the mutant proteins com- pared with the wild type. For the heat-inactivation experiments, LPH was incubated at 55°C for 45 minutes before assay; controls were incubated at 4°C.

Resu l ts

As expected, t ransfect ion of the fu l l - l eng th L P H

c D N A generates a h igh -mo lecu l a r -we igh t prote in , repre-

sent ing the unprocessed p r o L P H (Figure 1A, lane 3).


Table 1, Compar ison of Km Values for Lactase (Using Lactose as Substrate) and Phlorizin Hydro lase (Using ONP-glc as Subst ra te) Act iv i t ies of Rat Nat ive LPH Isolated From Microvi l lus Membranes and LPH Synthes ized by COS-7 Cells Transfected With the Wild-Type LPH Construct

Lactose ONP-glc Enzyme (mmol/L) (mmol/L)

Microvillus membrane LPH (3) 52 2.3 Wild-type LPH (3) 48 3.1

NOTE. The number of experiments is shown in parentheses.

Successful immunoprecipitation by the anti-LPH monoclonal antibody indicates that the antigenicity of the synthesized protein is similar to that of native LPH. Furthermore, comparison of K,~ values of immunoprecipi- rated COS cell LPH and rat microvillus membrane LPH gave comparable results, indicating that the substrate affinities were essentially the same in both COS ceils and native proteins (Table 1). Thus, the wild-type protein produced by the transfected cDNA is functionally identical to that produced by rat enterocytes. Similarly, Naim et al. 2° showed that a full-length human LPH cDNA that was transfected into COS cells generated functionally normal human LPH.

As shown in Table 2, the amino acid sequence around the glutamic acid identified as the active site nucleophile of domain III is identical in the three species that have been sequenced and is identical to the consensus active- site sequence for this family of glucosidases described by Trimbur et al. 21 The domain IV sequences are also identical in all three species. In this e ight-amino acid region, the only difference between the domain III and IV sites is a valine for isoleucine exchange. The high degree of

Table 2. Compar ison of Amino Acid Sequences Surrounding the Domain III and IV Act ive Si tes of Rat, Rabbit, and Human LPH

Source Domain Sequence

Rat

Rabbit

Human

III P IYIT~,I2V4NG

IV PIYVT~--Iv s °NG

III PIYITEI271NG

iV P IYVT~-I747NG

111 PIYITEI273NG IV PIYVTEI749NG

NOTE. Active-site glutamic acid is shown in bold. The superscripted number is the location of glutamic acid in the complete LPH sequence. The sequence PIYITENG is the consensus active-site sequence identified by Tdmbur et al. 21 for the family of glucosidases of which LPH is a member. 11 (Sequence data from Duluc et al, 9 and Mantel et al. 1°)

Table 3. Enzymatic Act iv i t ies of Transfected Rat Wild-Type and Mu tan t LPH Constructs Immunoprec ip i ta ted From COS-7 Ceils

Substrates

Lactose Cellobiose ONP-glc Enzyme (%) (%) (%)

Control (6) < 1 < 1 < 1 Wild type (6) 100 100 100 E1274D (6) 94 102 86 E1274G (6) 98 95 91 E1750D (3) < 1 < 1 26 E1750G (3) <1 < 1 24 Double (3) < 1 < 1 < 1 T1273P (3) < 1 < 1 < 1 Y1271F (3) 122 96 100

NOTE. Controls are cells that were processed identically except that no cDNA was transfected. All activities are expressed relative to that of the wild-type LPH, which is set at 100%. Lactose and cellobiose are substrates for the lactase, and ONP-glc is a substrate for phiorizin hydrolase. The double mutant has both E1274G and E1750G mutations in the same cDNA. The number of experiments is shown in parentheses.

sequence conservation suggests that any change may have dramatic effects. Indeed, by mutagenesis analysis of the structurally homologous glucosidase from Agrobacterium faecalis, Trimbur et al. 21 showed that every one of the nine substitutions for the glutamic acid nucleophile that they made, including aspartic acid, glutamine, aspara- gine, and glycine, reduced the activity of the enzyme by five orders of magnitude, indicating that the exact spatial relationship of the substrate and the nucleophile is critical.

When site-directed mutagenesis of the active-site nucleophiles of the full-length rat LPH was performed, the molecular mass and antigenicity of the synthesized proteins were not appreciably affected by the single amino acid substitutions (Figure 1k, lanes 4-6) . Unex- pectedly, the domain III mutations E1274D and E1274G had no detectable effect on the lactase or cellobiose activities of LPH (Table 3). The replacement of glutamic acid by aspartic acid only shortens the side chain of the putative nucleophile by one carbon and may not be expected to affect the activity dramatically. However, the complete elimination of the side chain by the glycine for glutamic acid substitution also had no effect on lactase or cellobiose activity. In contrast, when the phlorizin hydrolase activity was measured in these mutants, there was a consistent decrease in activity (Table 3). These data suggest that the domain III site accounts for none of the LPH lactase but for part of the phlorizin hydrolase activity.

It is likely that this is a specific result of the alteration of the nucleophile. Figure 1 shows that immunoprecipitation of both wild-type and mutant proteins is similar,


205kD-

l l 6 . 5 k D -

80kD-

49.5kD-

LPH

IgG

Figure 2. SDS-PAGE comparison of immunoprecipitated wild-type LPH and three mutant LPH proteins. LPH proteins were immunoprecipitated as described in Materials and Methods. Samples were electrophoresed on a 4% stacking and a 10% polyacrylamide separating gel. The gels were stained with Coommassie blue. The position of high- molecular-weight markers 205, 116.5, 80, and 49.5 kilodaltons (kD) are shown on the left. Lane 1, wild-type LPH; lane 2, E1274D nucleophile mutant LPH; lane 3, T1273P mutant LPH; and lane 4, Y1271F. Arrows indicate positions of the high-molecular-weight LPH precursor LPH and the immunoglobulin G (IgG) heavy chain.

suggesting no major differences in folding as a result of these mutations. Because the nucleophile itself is required for interaction with the substrate, it is unlikely that it is also required to maintain the structure of LPH. However, the highly conserved residues adjacent to the nucleophile may have a role in maintaining protein configuration. For example, when a T1273P mutant was assayed, no enzyme activity was detectable (Table 3). Proline residues are often sites of bending in proteins; therefore, this substitution might be expected to cause a change in protein configuration. In fact, when analyzed by SDS-PAGE (Figure 2), the antibody did not bring down any detectable protein, suggesting that this mutation sufficiently changes the protein conformation to eliminate antibody binding. On the other hand, the Y1271F mutation showed full activity and was effec- tively immunoprecipitated (Table 3 and Figure 2), indicating that removal of the hydroxyl group by a tyrosine to phenylalanine conversion had little or no effect on the structure of the LPH protein. Thus, recognition of the mutated LPH protein by the monoclonal antibody seems to be a sensitive indicator of structural changes in the protein.

In contrast to the domain III mutations, when the domain IV site was mutated, both lactase and cellobiose activities were undetectable in either the E1750D or E1750G mutants, although the immunoprecipitated proteins appeared identical to the wild type and domain

III mutants (Figure 1, lane 5). The domain IV mutants showed dramatically reduced (by about 75%) but not eliminated activity against substrates representing the phlorizin hydrolase specificity (Table 3) when expressed relative to the activity of the wild-type protein against the same substrate. Because the wild-type absolute activity against ONP-glc was considerably lower than activity against lactose, these mutants have very low enzyme activity. The E1750D and E1750G mutant LPHs consis- tently showed positive activity with the ONP-glc substrates at about 25% of the wild-type level, again suggesting that the unaffected domain III site, in fact, had a low level of activity against these substrates but not against lactose. These findings identify E1274 only as a phlorizin hydrolase site and E1750 as the lactase as well as a phlorizin hydrolase site. This result is the reverse of the identification of E1274 as the lactase nucleophile and E1750 as the phlorizin hydrolase nucleophile reported by Wacker et al. 8

Finally, the double mutant, which contained both E1274G and E1750G mutations, had no detectable activity against any of the substrates. This confirms that E1274 and E1750 represent the only LPH active sites in the protein and are essential to the activity of the enzyme.

Differential heat inactivation has been used by a number of investigators as a means of distinguishing two active sites in LPH. 3'<7 When the wild-type protein was

heated, lactase activity decreased by 82%, whereas the 0- nitrophenyl-~- 1,4-glucosidase activity decreased by 56% (Table 4), comparable in magnitude with previous re- ports of the differential heat stability of the activities. 4'7

Similarly, when the E1274G mutant was heated, lactase activity also decreased by 82%, whereas the ONP-glc activity decreased by 44%. The difference probably re- flects the elimination of the contribution of the domain III site. When the E1750G mutant, which has no lactase activity, was heated, the ONP-glc hydrolase activity due to the domain III site was reduced by 40%. Thus, the ONP-glc hydrolase activity of both sites was more resis- tant to heat inactivation than was the single lactase activity in domain IV. Furthermore, these data indicate that the previously reported inactivation profiles reflect only the greater lability of the single lactase site to heat, whereas the two separate phlorizin hydrolase sites are both comparatively less heat labile. Thus, heat inactivation does not permit distinction of the two active sites.

Discussion

These data confirm that E1274 and E1750 are essential for the enzyme activities of rat LPH. It has been suggested that the amino terminal domain I and


Table 4. Remaining Enzyme Activities of Rat Wild-Type and

Mutated LPH After Heat Inactivat ion

Substrates

Enzyme Lactose (%) ONP-glc (%)

Control <1 <1 Wild Wpe(3) 18 44 E1274G(3) 18 56 E1750G(3) ND 60

NOTE. Controls are cells processed identically except that no cDNA is transfected. Activities measured in unheated LPH were set at 100%. Lactose is a substrate for lactase, and ONP-glc is a substrate for phlorizin hydrolase. The number of experiments is shown in parentheses. ND, not done.

II portion of LPH, which is normally removed during processing in enterocytes, may show a previously uniden- tified glucosidase activity. 1° Based on sequence comparison with other hydrolases, Henrissat 1~ predicted that domains I and II would be noncatalytic because they do not contain glutamic acids in positions homologous to those in domains III and IV. This was confirmed by Wacker et al. 8 through their demonstration of labeled CBE binding to only rabbit E1271 and E1747. No evidence for enzymatic function of regions I and II has been reported. Assays of our double mutant show that this portion of the protein has no detectable activity against the substrates tested, which argues against the presence of any glucosidase activity similar to that shown by the domain III and IV sites. In contrast to the previous identification, s site-directed mutagenesis shows that E1750 is the lactase site nucleophile and is critical to the majority of the enzyme activity shown by rat LPH.

The equal precipitation of wild-type and mutant LPH from the transfected cells indicates that the altered nucleophile does not significantly affect the structure of the protein and the ability of the antibody to bind to it. The very different effects of insertion of a proline or a phenylalanine in the highly conserved region, which may have a structural function, adjacent to the nucleophile is consistent with this interpretation. Because the postu- lated mechanism of lactose hydrolysis by LPH requires that the substrate molecule interact with the active-site nucleophile, it is not surprising that modification of this amino acid apparently does not affect the structure of the enzyme. It is unlikely that the side chain required for substrate interaction is also required to maintain proper folding of the protein.

The high degree of sequence homology among all four domains led to the suggestion that LPH arose from an ancestral enzyme by two gene duplications. I° If so, it is likely that domain IV represents the ancestral ~-glucosi-

dase protein, as proposed by Gr~ibnitz e t a l . 22 Because lactase is confined to mammals, whereas the aryl-~-glucosidase or phlorizin hydrolase activity is reported to be present in all vertebrates, phlorizin hydrolase may be the phylogenetic precursor of LPH. 22 Thus, it is of interest and consistent with this hypothesis that, in rat, only domain IV has full enzyme activity, whereas the other domains show partial or no enzymatic activity. Of the enzyme sequences currently available, the sequence of LPH is most closely related to several bacterial glucosidases but to no other eukaryotic enzymes, n Thus, the evolutionary history of LPH remains obscure.

A somewhat similar situation has been described in mammalian hexokinase type I, an enzyme of 100 kilodaltons believed to have arisen from an ancestral protein of 50 kilodaltons by gene duplication and to have evolved distinct functions in the C-terminal and N-terminal halves. However, a recombinant protein consisting only of the 50-kilodalton C-terminal half has full function. 23 When the prosequence of human LPH, consisting of domains I and II of the protein with structural homology to domains III and IV, was removed, the protein synthesized after cDNA transfection remained intracellular, suggesting that it was necessary for transport of LPH to the cell surface. 24 Data from a study by Naim et al. 25

indicate that this 849-amino acid proregion of LPH may be required for proper folding of the mature molecule and, thus, is necessary for both transport to the cell surface and enzyme activity. These investigators transfected a cDNA from which the proregion had been removed and showed that, while a protein of the expected size was synthesized, most of the protein remained intracellular and, when immunoprecipitated, the protein had no detectable lactase activity.

The dramatic difference in substrate specificity of the two LPH active sites is remarkable because the regions immediately surrounding the nucleophile residues are highly conserved between the two domains as well as in all three LPH sequences (rabbit, human, and rat) so far reported, 9'1° as summarized in Table 2. A second region distal to each site is also highly conserved in LPH and other glucosidases. 2. The homologous region was shown to be required for full enzyme activity of the Agrobacterium glucosidase by Trimbur et al. 21 These investigators suggest that this region of the enzyme plays a role in substrate binding. Other regions of the protein or subtle differences in the im- mediate region of the active sites must be responsible for the striking difference in substrate specificity between domains III and IV. Determination of the structural basis of these substrate specificities will be the subject of future investigations.


References

1. Brady RO, Gal AE, Kanfer JN, Bradley RM. The metabolism of glucocerebrosides. II1. Purification and properties of glucosyl- and galactosyl-ceramide-cleaving enzyme from rat intestinal tissue. J Biol Chem 1965 ;240 :3766-3770 .

2. Kobayashi T, Suzuki K. The glycosylceramidase in the murine intestine. J Biol Chem 1981; 256 :7768-777 .

3. Leese H J, Semenza G. On the identity between the small intestinal enzymes phlorizin hydrolase and glycosylceramidase. J Biol Chem 1973 ;248 :8170-8173 .

4. Colombo V, Lorenz-Meyer H, Semenza G. Small intestinal phlorizin hydrolase: the ~-glycosidase complex. Biochim Biophys Acta 1973 ;327 :412-424 .

5. BOiler HA, van Wassenaer AG, Raghavan S, Montgomery RK, Sybicki MA, Grand RJ. New insights into lactase and glycosylceramidase activities of rat lactase-phlorizin hydrolase. Am J Physio] 1989; 257:G616-G623.

6. Birkenmeier E, AIpers DH. Enzymatic properties of rat phlorizin- hydrolase. Biochim Biophys Acta 1973 ;350 :100 -112 .

7. Lorenz-Meyer H, Blum AL, Haemmerli HP, Semenza G. A second enzyme defect in acquired lactase deficiency: lack of small intestinal phlorizin-hydrolase. Eur J Clin Invest 1972 ;2 :326-331 .

8. Wacker H, Keller P, Falchetto R, Legler G, Semenza G. Location of the two catalytic sites in intestinal ]actase-phlorizin hydrolase. J Biol Chem 1992 ;267 :18744-18752 .

9. Duluc I, Boukamel R, Mantel N, Semenza G, Raul F, Freund JN. Sequence of the precursor of intestinal lactase-phlorizin hydrolase from fetal rat. Gene 1991 ;103 :275-276 .

10. Mantel N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G. Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J 1988 ;7 :2705-2713 .

11. Henrissat BA. Classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991 ;280 :309 - 316.

12. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2rid ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

13. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain- terminating inhibitors. Proc Natl Acad Sci USA 1977 ;74 :5463 - 5467.

14. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985 ;82 :488 - 492.

15. Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzy- mol 1987; 154 :367-382 .

16. Kessler M, Acuto O, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Biochim Biophys Acta 1978 ;506 :136-154 .

17. B(Jller HA, Montgomery RK, Sasak WV, Grand R.I. Biosynthesis, glycosylation, and intracellular transport of intestinal lactase- phlorizin hydrolase in the rat. ,i Biol Chem 1987 ;262 :17026- 17211.

18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 ;227 :680-685 .

19. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biochem 1968 ;22 :99 -107 .

20. Naim HY, Lacey SW, Sambrook JF, Gething MJH. Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleared, enzymatically active, and transport-competent protein. J Biol Chem 1991 ;266 :12313-12320 .

21. Trimbur DE, Warren RAJ, Withers SG. Region-directed mutagenesis of residues surrounding the active site nucleophile in ~-glucosidase from Agrobacterium faecalis. J Biol Chem 1992; 267 :10248-10251 .

22. Gr~bnitz F, Seiss M, ROcknagel KP, Staudenbauer WL. Structure of the ~-glucosidase gene bglA of Clostridium thermocellum. Eur J Biochem 1991 ;200 :301 -309 .

23. Magnani M, Bianci M, Casablanca A, Stocchi V, Daniele A, AItruda F, Ferrone M, Silengo L. A recombinant human "mini"-hexokinase is catalytically active and regulated by hexose 6-phos- phates. Biochem J 1992 ;285 :193-199 .

24. Oberholzer T, Mantel N, Semenza G. The pro sequence of lactase- phlorizin hydrolase is required for the enzyme to reach the plasma membrane. FEBS Lett 1993 ;333 :127-131 .

25. Naim HY, Jacob R, Naim H, Sambrook JF, Gething M-JH. The pro region of human intestinal lactase-phlorizin hydrolase. J Biol Chem 1994; 269 :26933-26943 .

Received January 17, 1995. Accepted June 15, 1995. Address requests for reprints to: Robert K. Montgomery, Ph.D.,

New England Medical Center, Box 383, 750 Washington Street, Bos- ton, Massachusetts 02111. Fax: (617) 636-4233.

Supported by grant R01 DK32658 from the National Institutes of Health; grant P30 DK34928 from the National Institutes of Health Digestive Disease Core Center, NEMCH; The Netherlands Digestive Diseases Foundation, Nutricia, The Netherlands; the Foundation De Drie Lichten; the Spinoza Funds; Mead Johnson; and a NATO Collabo- rative Research Grant.

The authors thank Dr. Alan Kopin, New England Medical Center, for advice on the site-directed mutagenesis procedure; Dr. Jane G. Schaller, Pediatrician-in-Chief, for valuable support; and Drs. Jelle Haringsma and Paul Matsudaira for participation in early aspects of this project.

verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis

Documents