Human lactase and the molecular basis of lactase persistence

Biochemical Genetics, Vol. 23, Nos. 5/6, 1985

Human Lactase and the Molecular Basis of Lactase Persistence

Jennifer Potter, l M a c - W a n Ho, 1'2 Hilary Bolton, 1 Anna J. Furth, 1 Dallas M. Swallow, 3 and Beatrice Grifliths 3

Received 15 Nov. 1984--Final 4 Jan. 1985

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in Mr (16OK). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl-~-galactoside and fl-glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with laetase under all conditions investigated," coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.

KEY WORDS: human lactase; purification; monoclonal antibody; polyclonal antibodies; immu- nology of lactase persistence.

Financial support was provided by the Nuffield Foundation, the Medical Research Council, and the Open University Research Committee Fund.

Biology Discipline, Open University, Walton Hall, Milton Keynes, MK7 6AA, U.K. 2 To whom correspondence should be addressed. 3 MRC Human Biochemical Genetics Unit, University College, London, NW 1 2HE, U.K.

423 0006-2928/85/0600-0423504.50/0 © 1985 Plenum Publishing Corporation

424 Potter, Ho, Bolton, Furth, Swallow, and Griffiths

INTRODUCTION

Lactase (EC 3.2.1.23.62) is found exclusively in the small intestine and, in almost all mammals, only during the perinatal stages of development. The notable exceptions are North Europeans and isolated African tribes, where a high proportion of individuals has lactase persisting into adult life (Dahlqvist, 1977). The genetics of lactase persistence is as yet poorly understood. It is commonly supposed that some regulatory gene mutation is involved which prevents the normal switch-off in enzyme synthesis on weaning (e.g., Kretchmer, 1977). Ho et al. (1982) demonstrated a gene-dosage effect among putative homozygous persistent, homozygous nonpersistent, and het- erozygous individuals by means of the ratio of sucrase to lactase activities, thus suggesting a cis-acting regulatory mechanism--if, indeed, a regulatory gene is involved.

We have now purified the enzyme from adult British natives by a combination of conventional biochemical techniques and monoclonal immunoadsorbent chromatography. The results of the purification and investigations of the immunological and other properties of the enzyme in relation to the molecular basis of lactase persistence are reported in this paper. Skovbjerg et al. (1981) have purified lactase by a different route involving conventional immunoadsorbent chromatography after three successive rounds of immunization. We have extended their immunological investigations (Skovbjerg et al., 1978) of adult hypolactasia to infant lactase as well as lactase from different human populations.

MATERIALS AND METHODS

Materials

The source of the enzyme was postmortem lower jejunum (Ho et al., 1982). Special chemicals and reagents used were as follows. Lactose (glucose free), cellobiose N-palmitoyl-dihydroglucocerebroside, glucose oxidase (type V from Aspergillus niger), papain (twice crystallized), trypsin (type III), Emulphogen, o-dianisidine, adenosine triphosphate (ATP), agarose, p-chloromercuriben- zoate (PCMB), phenyl-methyl-sulfonyl-fluoride (PMSF), L-leucyl-L-alanine, L-leucyl-L-leucyl-L-leucine, L-phenylalanyl-L-tyrosine, L-phenylalanyl-L-proline, L-alanyl-p-nitroanilide, p-nitroaniline, L-methionine, snake venom (Cro- talus adamantus), carbonic anhydrase and myosin [29K and 200K molecular weight markers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)], biotin concanavalin A (Con A) and avidin peroxidase were from Sigma (St. Louis, Mo.). 4-Methylumbelliferyl derivatives of/3-galactose (4MUgal), ~3-glucose (4MUglu) and phosphate, and 4-methylumbelliferone were from Koch Light Laboratories Ltd. (Colnbrook, Bucks., U.K.) /3-D-

Human Lactase 425

Galactose dehydrogenase (purified from Pseudomonas, 5 mg/ml), hexokinase/ glucose-6-phosphate dehydrogenase (purified from yeast, 15 mg/ml), soybean trypsin inhibitor, NAD, NADP, horseradish peroxidase (grade II), and molecular weight markers for SDS-PAGE (myoglobin, 17K; glyceraldehyde- 3-phosphate dehydrogenase, 36K; phosphoglycerate kinase, 47K; pyruvate kinase, 60K; phosphorylase a, 94K; RNA polymerase, 160K; and thyroglobu- lin, 340K) were from Boehringer-Mannheim (Lewes, East Suffolk, U.K.). Triton X-100 (B grade) and sodium taurocholate (A grade) were from Calbiochem (La Jolla, Calif.); Sepharose 4B, CNBr-Sepharose 4B, and Sephacryl $300 were from Pharmacia (Uppsala, Sweden). Protein A Bacte- rial Adsorbant was from Miles Laboratories (Slough, U.K.), nitrocellulose sheets were from Bio-Rad (Watford, Herts., U.K.), 125I was from Amersham (Bucks., U.K.), phlorizin was from Fluka (Bucks, Switzerland), and complete Freund's adjuvant was from Difco (Detroit, Mich.). Concanavalin A (Vector Labs), maltose (extra pure), and Aristar grades of sucrose, glucose, and galactose were obtained from BDH Chemicals Ltd. (Essex, U.K.).

Tissue Extracts

Brush-border membrane extracts were prepared according to a modification of Louvard et al. (1973). Postmortem frozen jejunum was thawed, weighed, slit open, and washed in ice-cold buffer (0.05 M Tris-HCl, pH 7.3, with 0.15 M NaC1, 10 mN MgCI2). The mucosa, scraped off with a glass microscope slide, was suspended in six times its weight of the same buffer to which 1 mM CaC12 and 0.25 M sucrose were added. This was sonicated (MSE Soniprep 150; amplitude, 22 U) for 5 x 10-sec bursts (10 sec between each burst). Large debris were removed by spinning at 10,000g for 15 rain. The brush-border membrane was pelleted at 105,000g for 30 rain, and the pellet extracted with 6 vol of 0.1 M sodium barbitone buffer, pH 8.0, containing 2% Emulphogen for 30 rain at room temperature. The insoluble residue was removed by centrifugation.

Total membrane extracts were made as follows. Postmortem jejunum was cut into small pieces while still frozen and homogenized in 2 vol of distilled water. After centrifugation at 40,000g for 60 rain, the pellet was rehomogenized in 1 vol of 0.1 M sodium phosphate buffer, pH 8.0, containing 2% Triton X-100 and left at 4°C for 30 min. The insoluble residue was removed by centrifugation.

Enzyme Assays

In all assays, 100 #1 of buffer-substrate solution was added to 10 or 20 #1 of enzyme solution. Lactase, sucrase, and maltase were assayed with the natural substrates as described previously (Ho et al., 1982). Cellobiase was assayed at


a substrate concentration of 20 mM in the buffer used for lactase and sucrase (0.05 M sodium acetate, pH 5,6, 10 mM EDTA); the reaction was stopped by boiling for 1 min, and the liberated glucose measured with the glucose oxidase/peroxidase method (Ho et al., 1982). The hydrolysis of MUgal and MUglu (1 and 2 mM, respectively) was done in the same buffer used for lactase to which 0.5 mM PCMB was added to inhibit lysosomal and other nonspecific /3-galactosidases (Ho and O'Brien, 1971). The reaction was stopped with 3 ml of 0,1 M glycine-NaOH, pH 10.5, and the fluorescence read at 450 nm (365-nm excitation) against a standard curve for 4-methylumbelliferone. Phlorizin hydrolase was assayed in one step at a substrate concentration of 1 mM in 0.1 M triethanolamine-HC1, pH 7.4, with MgSO4 (4 mM), ATP (2 mg/ml), NADP (0.8 mg/ml), and hexokinase/glucose-6-phosphate dehydrogenase (7/zl/ml). The reaction was stopped with 1 ml of 0.1 N NaOH, and the absorbance read at 365 nm against a standard curve for glucose. Glucocerebrosidase was assayed at 0.17 or 1.7 mM glucocerebroside in the buffer systems for phlorizin hydrolase or lactase or in the buffer systems specifically designed for glucocerebrosidase by Ho et al. (1973) and Leese and Semenza (1973), respectively. The glucose released was measured as for phlorizin hydrolase. Alkaline phosphatase was assayed with 1 mM methylumbelliferyl-phosphate in 0.1 M Tris-HC1, pH 8.8. After 5 to 15 min of incubation at 37°C, 3 ml of 0.1 M glycine-NaOH, pH 10.5, was added and the fluorescence read immediately. Aminopeptidase activity was assayed with 1 mM L-alanyl-p-nitroanilide in 0.05 M Tris-HC1, pH 7.3. After 30 rain at 37°C, the reaction was stopped with 2 ml glycine-NaOH, pH 10.5, and the absorbance read at 380 nm against a standard curve for p-nitroaniline. Various peptidases were assayed with leucyl-alanine, phenylalanyl-proline, and phenylalanyl-tyrosine, and tripeptidase with leucyl-leucyl-leucine, all at 1 mM in 20 mM sodium phosphate, pH 7.4, containing 1 mM MnC12, 0.1 mg/ml peroxidase, 0.05 mg/ml o-dianisidine, and 0.3 mg/ml snake venom (modified from Harris and Hopkinson, 1976). After 5-15 min at 37°C, the reaction was stopped with 1 ml of 0.1 N NaOH/0.1% Triton X-100, and the absorbance read at 425 nm against a standard curve for L-methionine. Protein was measured by the method of Lowry et al. (1951).

Protease Treatments

Papain was used at approximately 1 part papain to 10 parts (by weight) protein in 0.1 M sodium phosphate, pH 6.0, containing 0.15 mg/ml cysteine-- HC1. After 1 hr at 37°C, the reaction was stopped by adding PCMB to a final concentration of 0.5 mM. Trypsin treatment [1 part trypsin to 10 parts (by weight) protein] was carried out in 0.05 M Tris-HC1, pH 8.0, at 37°C. After 1 hr the reaction was stopped by adding an amount of soybean trypsin inhibitor equal to that of trypsin.

Human Lactase 427

Electrophoresis

Starch gel electrophoresis and enzyme staining were as described by Ho et al.

(1982) and Harris and Hopkinson (1976). SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) was performed in 4-15% acrylamide at 45 mA (room temperature) in a discontinuous buffer system (Laemmli, 1970). Protein was stained with Coomassie brilliant blue.

Glycoprotein Staining

This was done according to Karlsson et al. (1983), with 125I-Con A binding and autoradiography (2 to 5 days' exposure with Kodak RPRoyal X-Omat film), or according to a modification of Gordon-Weeks and Harding (1983) on electroblotted proteins after SDS-PAGE.

Partial Purification of Lactase

A brush-border membrane extract (2 ml) was applied to a Sephacryl $300 column (2.5 x 85 cm) at 4°C and eluted with 10 mM sodium phosphate, pH 8.0/1% Emulphogen at 200 ml/hr. Protein was monitored by absorbance at 280 nm. Fractions of 3 ml were collected and assayed for lactase and sucrase. Active fractions were pooled so as to minimize overlap between the two activities and concentrated by ultrafiltration (Amicon).

Immunization for Polyclonal Antibodies to Lactase

Rabbits were bled for control sera and then immunized with 200 #g partially purified or purified lactase in complete Freund's adjuvant subcutaneously. Another injection was given in 1 month's time, followed by intravenous injection (in phosphate-buffered saline) 2 weeks later. The rabbits were bled in another 2 weeks. Boosters were then given every 4 weeks and antisera collected 2 weeks after each injection. Pooled sera were mixed with an equal volume of saturated Na2SO4. The precipitate, which contained all the antilactase activity, was redissolved in a small volume of phosphate-buffered saline and dialyzed against the same.

Crossed Immunoelectrophoresis and Immunodiffusion

Crossed immunoelectrophoresis was done as described by Skovbjerg et al.

(1980) in 1% agarose. Immunodiffusion was performed in 1% agarose gel in phosphate-buffered saline containing 0.2% Triton X-100. Precipitin arcs for lactase, phlorizin hydrolase, and cellobiase were visualized by staining as for starch gel electrophoresis.

428 Potter, Ho, Bolton, Furth, Swallow, and Grifllths

lmmunotitration Tests

Antigen and antibody solutions were mixed in a total volume of 100 ul phosphate-buffered saline/0.2% Triton X-100 and left overnight at 4°C. After centrifugation at 17,000g for 30 rain, the supernatant was assayed for lactase activity.

Purification of Lactase by Monoclonal lmmunoabsorbent Chromatography

Preparation of the monoclonal antibody to lactase is described elsewhere (Swallow et al., 1985). The monoclonal antibody, partially purified by precipitation at 50% saturation of ammonium sulfate from pooled hybridoma cell media, was coupled to CNBr-Sepharose 4B according to the instructions of the manufacturer. The resultant immunoadsorbent gel was packed into a column (1.5-cm diameter, 1- to 10-ml total volume) and equilibrated with binding buffer (25 mM sodium phosphate, pH 7.5/0.15 M NaC1/0.2% Triton X-100/10 mM EDTA). The total membrane extract, diluted 10-fold with the binding buffer, was applied to the column at a flow rate of about 2 column vol/hr. The applied sample was allowed to recirculate with an LKB perpex pump for 30 rain at room temperature, then another 17 hr at 4°C. The total breakthrough fraction was collected. The column was then washed with 20 or more vol of binding buffer in the cold, followed by 1 column vol of the buffer from which sodium phosphate was omitted. The column was allowed to warm up to room temperature, and lactase was rapidly eluted under gravity within 1 to 3 column vol of elution buffer (0.1 M sodium acetate, pH 3.5/0.15 M NaC1/0.2% Triton X-100/1 mM EDTA).

RESULTS

Partial Purification of Lactase

Figure 1 gives Sephacryl $300 elution profiles of lactase and sucrase and the SDS-PAGE of the pooled and concentrated fractions: a 13-fold purification over the crude homogenate is achieved in the brush-border membrane extract; subsequent gel filtration results in a further 4-fold purification. On SDS- PAGE, the pooled fractions containing lactase show a single polypeptide (M, 160K) when stained with Coomassie blue (Fig. lb). ~25I-Con A staining, which is much more sensitive, gives at least eight other glycoprotein bands (Fig. lc). The specific activity of lactase in the preparation (pooled fractions 60 and 61) was 5.9 U/rag. This preparation was used to raise both monoclonal and polyclonal antibodies.

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Fig. 1. Gel filtration of brush-border membrane extract on Sephacryl $300. (a) Elution profiles: protein ( . . . . ), lactase (©), and sucrase (e). (b) SDS-PAGE stained with Coomassie blue. (c) SDS-PAGE stained with '25I-Con A. Unlabeled lane, applied extract; lanes 1-5, successive pooled and concentrated fractions--58 and 59, 60 and 61,62 and 63, 64 68, and 69-76.

Purification of Lactase by Monoclonal Immunoadsorbent Chromatography

The results of a typical run are summarized in Table I. The final purification is approximately 1000-fold over the crude homogenate; and the specific activity, 36.6 U/mg protein [a value of 32 U/mg was reported by Skovbjerg et al. (1981)]. The elution profiles of lactase, phlorizin hydrolase, and meth-

430 Potter, Ho, Bolton, Furth, Swallow, and Grifliths

Table I. Purification of Lactase by Immunoadsorbent Chromatography

Total protein Sp act R e c o v e r y Purification Step (mg) ° (U/mg) (%) (fold)

Homogenate 768.00 0.031 100 1 Triton extract 200.00 0.108 91 3 Column eluate 0.17 36.600 25 1161

aThe figures are calculated per 10 g wet weight of jejunum.

ylumbelliferyl-galactosidase (Fig. 2a) are all concordant, indicating the presence of all the activities on a single enzyme species. The SDS-PAGE of the proteins in different fractions shows that the purified enzyme is a single polypeptide (Mr 160K) on both Coomassie blue and 125I-Con A staining (Fig 2b). It is essentially free of all dipeptidases and tripeptidases (limit of detection, 0.008 U/nag), aminopeptidase (limit of detection, 0.002 U/rag), sucrase and maltase (limit of detection, 0.008 U/rag), and alkaline phosphatase (limit of detection, 0.002 U/mg).

Soluble and Membrane-Bound Forms of Lactase

In a homogenate made in the presence of detergent, lactase activity is distributed in varying proportions between the cytosol and the total membrane fractions. The proportion of soluble activity increases with the storage time of the postmortem material at -20°C suggesting that the membrane-bound form is converted to the soluble form by endogenous proteolytic activity. The membrane-bound (amphiphilic) lactase can be extracted completely by 1 to 3% Triton X-100 or Emulphogen. Starch gel electrophoresis reveals that the predominant species in the cytosol and in the membrane (detergent extracted) exhibit widely different mobilities. The membrane amphiphilic form fails to enter the gel unless detergent is incorporated in the matrix (Fig. 3). Treatment of the amphiphilic form with papain or trypsin converts it to a species resembling the soluble form. The mobilities of the soluble and protease- derived forms are very similar and are not affected by the presence or absence of detergent in the gel matrix. These findings indicate that the amphiphilic lactase possesses a hydrophobic moiety--cleaved off by the proteases--which is capable of interacting with the detergent micelles and which is probably responsible for anchoring the enzyme to the membrane in vivo.

Subunit Structure of Lactase

The detergent-extracted amphiphilic lactase, purified on immunoadsorbent chromatography, retains its typical mobilities on starch gel electrophoresis with and without Triton X-100 (see above). It has a M~ of 160K on

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Fig. 2. Monoclonal immunoadsorbent chromatography. (a) Elution profiles: protein ( . . . . ), lactase (O), MU-fl-galactosidase (O), and phlorizin hydrolase (×). Total column volume is 1 ml. B, breakthrough unadsorbed fraction; W, wash buffer applied; E, elution buffer applied. (b) SDS-PAGE of fractions from a 10-m] column, stained with lZS1-Con A. Lane T, applied total membrane extract; lane 1, unadsorbed; lanes 2-8, successive wash fractions; lanes 9-19, successive eluted fractions of lactase. The peak of lactase activity is indicated by the arrow.

432 Potter, Ho, Bolton, Furth, Swallow, and Grifliths

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Fig. 3. Starch gel electrophoresis of soluble and detergent- extracted membrane-bound form of lactase with and without protease treatment (a) Ordinary starch gel; (b) starch gel with 1% Triton X-100. Lane 1, detergent extract of membrane, trypsin treated; lane 2, cytosol fraction, trypsin treated; lane 3, detergent extract of membrane untreated; lane 4, cytosol fraction, untreated, d, detergent-extracted membrane form; s, soluble cytosol form. Replacing trypsin with papain gives the same results.

SDS-PAGE under standard denaturing conditions (boiling in 1% SDS, 2% mercaptoethanol) (Fig. 4a, lane 2). When mercaptoethanol is omitted, and the enzyme boiled in 1% SDS, a band of approximately 320K is seen in addition to the 160K band (Fig. 4a, lane 4). Under nondenaturing conditions (0.1% SDS, no mercaptoethanol and no boiling), a single species with an apparent Mr of about 290K is seen (Fig. 4a, lane 6). These observations suggest that "native" lactase is a dimer of subunits equal in Mr, which is fully dissociated by boiling in the presence of SDS and mercaptoethanol. Only partial dissociation is observed when the enzyme is boiled in the presence of SDS without mercaptoethanol. The difference in apparent Mr between the denatured and the nondenatured dimers (Fig. 4a, lanes 4 and 2) may simply reflect a difference in conformation. However, the relative mobilities of undenatured proteins on SDS electrophoresis are not necessarily related to Mr in a simple way.

Trypsin treatment reduces the apparent Mr of the native enzyme as well as the denatured dimer by a small amount (between 5 and 10K) (compare lanes 5 and 6 and lanes 3 and 4 in Fig. 4a). There is also a corresponding change in mobility in the 160K subunit (which is detectable by a careful alignment of the 125I-Con A autoradiograph over a horizontally lined paper). In order to demonstrate the effect of papain and trypsin on the 160K subunit with greater precision, a more sensitive staining technique was used, which depends on biotinylated Con A binding to electroblotted SDS-PAGE. This system also gave a better resolution of the bands. Figure 4b shows that papain and trypsin both reduce the apparent Mr of the 160K species by about 5000.

Human Lactase 433

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Fig. 4. Investigations on subunit structure of purified lactase using protease treatments. (a) SDS-PAGE stained with ~25I-Con A. Lane 1, trypsin treated, boiled with 1% SDS and 2% mercaptoethanol; lane 2, untreated and boiled as in i; lane 3, trypsin treated, boiled with 1% SDS; lane 4, untreated and boiled as in 3; lane 5, trypsin treated, incubated for 1 5 min at room temperature with 0.1% SDS; lane 6, untreated and incubated as in 5. (b) Electroblotted SDS-PAGE stained with peroxidase-coupled Con A. All samples were boiled with 1% mercaptoethanol and 2% SDS. Lanes 1-3, untreated, papain treated, and trypsin treated, respectively; similarly for lanes 4-6 and lanes 7-9. Relative loading of protein: 1 3, 3.5; 4 6, 2; and 7-9, 1.


At the heaviest loading (lanes 1-3), a cross-linked dimer (Mr 320K) not dissociated by boiling in SDS/mercaptoethanol is detected in the control. [We have previously identified this cross-linked species exclusively in the microvil- lar membranes fraction of adult human jejunal enterocytes (Bolton et al., 1985).] This is completely absent after papain treatment, whereas after trypsin treatment a faint band of Mr 305K is present instead (lane 3),

These changes in apparent Mr after protease treatments are correlated with alterations in mobilities on starch gel electrophoresis described above (Fig 3), namely, the untreated enzyme migrated as the membrane form and the treated enzyme migrated as the soluble form. These findings suggest that a small peptide (about 5000) is removed from both subunits in the dimer. As protease treatment also destroys the ability of the enzyme to bind to detergent micelles, the peptide removed probably contains the hydrophobic anchor to the cell membrane in vitro.

The visualization of both the amphiphilic and the hydrophilic forms of lactase by Con A binding shows that the enzyme is a mannose-containing glycoprotein.

Substrate Specificities of Lactase

These studies were carried out mostly on the brush-border membrane extracts. The kinetic constants for different substrates are given in Table II. The Km for lactose is significantly affected by the assay buffer. In our system (sodium acetate, p H 5.6), the Km is about 3.6 raM, whereas that in citrate phosphate, p H 6.0 (used in most other studies), is 13.3 raM. The constants for MUgal and MUglu are very similar to each other and to those for lactose, indicating that the active site recognizes the/3-glucoside and the/3-galactoside moiety equally. Cellobiose is hydrolyzed at a significantly lower rate than lactose, although the Km is five times smaller. The Km for phlorizin is at least an order of magnitude lower than those for the other substrates. Glucocere-

Table II. Kinetic Constants for Lactase Using Different Substrates

Substrate Km (mM) a Relative Vm~x

Lactose 3.57 b (4) 100 13.33 c (2) 101

MUglu 3.52 b (2) 91 MUgal 3.85 b (I) 122 Cellobiose 2.20 c (2) 15 Phlorizin 0.14 c (3) 18 Glucocerebroside - - (3) 0

aThe values are the average for the number of separate bAcetate buffer, pH 5.6; see text. cCitrate phosphate buffer, pH 6.0; see text.

determinations given in parentheses.

Human Lactase 435

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b r o s i d e t u r n s ou t no t to b e a s u b s t r a t e for l ac ta se . N o ac t i v i t y cou ld be

d e m o n s t r a t e d in e i t h e r t h e b r u s h - b o r d e r m e m b r a n e e x t r a c t or t h e pur i f i ed

l a c t a s e p r e p a r a t i o n s in a n y of t h e b u f f e r s y s t e m s t e s t ed . G l u c o c e r e b r o s i d a s e

ac t i v i t y cou ld b e d e t e c t e d in t h e t o t a l m e m b r a n e e x t r a c t of t h e h u m a n sp leen

on ly in t h e bu f f e r s y s t e m of r i o et al. ( 1 9 7 3 ) .

Table Ill. Precipitation of Lactase by Antibody Preparations Absorbed with Different Extracts a

Antibody preparation Enzyme activity precipitated (%)

Control unabsorbed 100 Absorbed with equivalent adult

positive extract b 5 Absorbed with equivalent infant

extract b 7 Absorbed with British adult

negative extract c 82 Absorbed with West Indian adult

negative extract c 90

aThe antibody was mixed with the absorbing extract in the usual immunotitration buffer and left overnight at 4°C. After centrifugation, the supernatant was transferred to a fresh tube and heated for 30 min at 50°C (to inactivate any remaining lactase activity) before testing against a fixed amount of a standard lactase extract.

bThe antibody was absorbed with a previously determined amount of extract sufficient to precipitate all or nearly all of the antibody.

c The amount of absorbing extract used contained the same amount of protein as the adult positive extract.


Fig. 6. Crossed immunoelectrophoresis of lactase. (a) Adult persistent (lactase activity positive; (b) infant; (c) adult nonpersistent (lactase activity negative). All were stained for lactase activity.

Immunological Properties of Lactase and the Molecular Basis of Lactase Persistence

The immunotitration characteristics of adult and infant lactase are very similar (Fig. 5). Equal activities precipitate at the same antibody concentra- tions. Individuals lacking lactase activity (lactase-nonpersistent homozygotes) also do not have appreciable cross-reacting material as demonstrated by absorption studies (Table III).

On crossed immunoelectrophoresis (Fig. 6), sizeable precipitin peaks staining for lactase are present in both adult and infant extracts, whereas lactase-nonpersistent individuals give very small peaks. The lactase peak in all cases stains for phlorizin hydrolase (as well as for cellobiase). The reduction in lactase activity in nonpersistent individuals is accompanied by the corresponding reduction in phlorizin hydrolase activity measured both by enzyme assays in crude extracts and by quantification on crossed immunoelectrophoresis (see Fig. 6). The observations are so far compatible with the presence of both phlorizin hydrolase and lactase activities on a single protein species. This is confirmed on immunodiffusion using a polyclonal antibody preparation raised against pure lactase from the monoclonal immunoadsorbent column (Fig. 7). Lactase and phlorizin hydrolase activities stain coincidentally with each other and with the precipitin arcs (stained with Coomassie blue) (Figs. 7a-c). There

Human Lactase 437

2

4

Fig. 7. Immunodiffusion of lactase and phlorizin hydrolase. (a-c) Identical plates stained for protein (a), lactase (b), and phlorizin hydrolase (c). Antilac- tase antibody was in the center well. Wells 1 to 5 contained total membrane extracts from lactase- persistent adult N5297, lactase-nonpersistent adult N4649, infant N5296, lactase-persistent adult N5019, and infant N5088, respectively. Well 6 contained the soluble cytosol fraction from lactase-persistent adult N5297. (d) Immunodiffusion plate stained for protein only. Antilactase antibody was in the centre well. Wells 1 to 6 contained total membrane extracts from lactase-persistent adult PM270, lactase-nonpersistent adult N5143, lactase-nonpersistent adult N4091, lactase-persistent adult PM272, lactase-persistent adult Asian Indian, and infant N5088, respectively. (a-c) Stained in washed, unpressed gel plates while still hydrated; (d) stained after pressing to remove excess liquid and then air-dried.

is, moreover, a complete fusion of precipitin arcs between adult and infant extracts (Figs. 7a and d), as well as between adult British native extracts and an extract from a lactase-positive adult Asian Indian (Fig. 7d). This is consistent with the similarity between infant and adult lactase indicated by the immunotitration tests (Fig. 5) and also suggests the absence of an immunological difference among lactase proteins in different human populations. Extracts from individuals of the adult British native population lacking lactase activity (nonpersistent homozygotes) also lack precipitin arcs in all


cases examined (Figs. 7a and d), indicating that enzymically inactive protein is absent.

DISCUSSION

By means of monoclonal immunoadsorbent chromatography, a rapid proce- dure for purifying lactase is achieved, which gives a purer product at a higher recovery rate than the previous method of Skovbjerg et al. (1981) using polyclonal immunoadsorbent chromatography.

Our investigations on the subunit structure of the purified enzyme also suggest that both the subunits of lactase--which are the same in apparent molecular mass--possess a hydrophobic anchoring peptide that is cleaved off by papain or trypsin. This raises the question concerning the identity of the lactase-phlorizin hydrolase complex, to which the two subunits are presumed to correspond (Semenza, 1981). Thus far, no definitive evidence has been produced that the two subunits of lactase are different. On the contrary, our own results are compatible with the alternative interpretation. The subunits are indistinguishable in M~ both before and after protease treatment (see Fig. 4). Moreover, phlorizin hydrolase is associated with lactase activity under all conditions investigated. The two activities coelute on immunoadsorbent chromatography, as well as on gel filtration under both dissociating and nondissociating conditions (Bolton et al., 1983). Immunoelectrophoresis gives a precipitin peak in infants and adults which stains for both activities. In adults in which lactase is nonpersistent, phlorizin hydrolase activity is also much reduced, and the lactase/phlorizin hydrolase peak on immunoelectrophoresis correspondingly diminished (Fig. 6). On immunodiffusion, phlorizin hydrolase stains coincidentally with lactase and with the single precipitin arc present in both adult and infant extracts; in lactase-nonpersistent extracts, no precipitin arc is formed, and correspondingly, no lactase or phlorizin hydrolase activity is stained (Fig. 7).

In our immunological investigations, we have demonstrated the absence of an immunological difference between infant and adult lactase. This, together with the corresponding reduction of antigenic material in individuals with little or no lactase activity persisting, strongly suggests that lactase persistence is due to the continued synthesis of the infant enzyme which normally switches off on weaning.

ACKNOWLEDGMENTS

We are grateful to Dr. Sue Povey for her continuing interest, support, and helpful discussions. Thanks are also due to Fiona Green for help with the

Human Lactase 439

tissue culture and to Steve Walters, Dawn Sadler, and Tina Davies for immunizations and collection of antisera. Autopsy materials were kindly provided by the Pathology Departments of Peterborough District Hospital, John Radcliffe Hospital of Oxford, and University College Hospital, Lon- don.

REFERENCES

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Human lactase and the molecular basis of lactase persistence