lactase; origin, gene expression, localization, and function

Pergamon

Nutrition Research, Vol. 14, No. 5, pp. 775-797, 1994 Copyright @ 1994 Elsevier Sciear.e Ltd Printed in the USA. All rights reserved

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LACTASE; ORIGIN, GENE EXPRESSION, LOCALIZATION, AND FUNCTION

Edmond H.H.M. Rings, M.D. 1", Erik H. van Beers, M.Sc. 1, Stephen D. Krasinski, Ph.D. 2, Menno Verhave, M.D. 2, Robert K. Montgomery, Ph.D. 2, Richard J. Grand, M.D. 2, Jan Dekker, Ph.D. 1, Hans A. BOiler, M.D., Ph.D. 1.

Divisions of Pediatric Gastroenterology and Nutrition, Departments of Pediatrics~ IAcademic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and The Boston Floating Hospital for Children, New England Medical Center Hospitals, Tufts University School of Medicine, Boston, MA, USA.

ABSTRACT

Lactase-phlorizin hydrolase is a small intestinal enzyme responsible for the hydrolysis of the carbohydrate lactose in mammalian milk. During the neonatal period the enzyme is crucial for the nutrition of humans and most other mammals. Subsequently, the specific activity of lactase decreases to low adult levels. In most adult humans and other mammals, large amounts of lactose are no longer tolerated, and lactose ingestion may lead to gastrointestinal symptoms. People of Caucasian extraction and a few isolated other groups form a clear exception with regard to this pattern; high levels of lactase activity are maintained, enabling these people to consume dairy products during adult life. This review will describe the role of lactase in the digestion of lactose in mammalian milk. The function and origin of the enzyme will be outlined, and the review will examine relevant issues regarding the consumption of lactose and the clinical syndrome of lactose intolerance. Furthermore, insight provided by molecular and cell biology into gene structure, promoter function, gene transcription, localization of expression in the small intestine, biosynthesis of lactase protein, and enzyme specificities will be outlined. Current thinking with respect to the regulation of expression of lactase during development, and the differences in expression between species and different human populations will also be discussed.

Key words: Beta-galactosidases; Beta-glucosidases; Lactase-phlorizin hydrolase; Lactose intolerance; Milk.

Address for correspondence: Edmond H.H.M. Rings, Division of Pediatric Gastroenterology and Nutrition, G8-260, Department of Pediatrics, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

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776 E.H.H.M. RINGS et al.

INTRODUCTION

What is lactase and where does i t come f r om?

Carbohydrates form an essential part of the human diet, and are present as monosaccharides, disaccharides, or complex polymers of saccharides. Quantitatively, the most common polymeric carbohydrates are starch (plant origin), glycogen (animal origin), and cellulose (plant origin). The predominant forms of free disaccharides are sucrose (plant origin) and lactose (animal origin). These carbohydrates are degraded by a limited number of enzymes, whose specificities are closely related to the chemical bonds between the monosaccharide consti tuents (1). The majori ty of the chemical bonds in the food-based carbohydrates are of the a-configuration. However, this review will concent ra te on the small intestinal enzyme lactase-phlorizin hydrolase, which is capable of hydrolyzing B-linked disaccharides of glucose and galactnse. Subsequently, we shall refer to this enzyme simply as lactase. Lactase is especially crit ical for the newborn mammal, because this enzyme hydrolyzes the B-linked disaccharide in mammalian milk, lactose. Lactose forms the primary carbohydrate source in the diet of the newborn.

Lactase is a type I t ransmembrane glycoprotein and contains both B-galactosidase and B-glucosidase activities within one polypeptide. Based on sequence similarities, human lactase belongs to a supei'family of B-glycosidases comprising both B-glucosidases and B-galactosidases as found in archaebacteria , eubacteria, and fungi (2,3,4). This sequence similarity is exemplified by the B-glucosidase (cellobiase) from Clostridium thermoceHum. This 51 kDa enzyme shows extensive homology to human lactase (5). Lactase itself exhibits four homologous regions, each representing a potential molecular mass of about 50 kDa. The C. thermoceHum B-glucosidase resembles sequences in each of the four regions of mammalian lactase, providing support for the idea that the mammalian lactase gene has evolved through an evolutionary process involving two subsequent duplications of an ancestral gene (6,7). This four-fold internal homology, which occurs in lactase of all mammals studied thus far (i.e. human, rabbit and rat), suggests the presence of conserved functional domains. These homologous regions in each lactase molecule have been designated region I through IV (Fig. 1 ). The potential N-glycosylation sites are largely conserved among the di f ferent species (Fig. 1 ). However, between regions, N-glycosylation sites vary, suggesting region-specific functions (7). Furthermore, the lactase sequences of the three species show very similar t ransmembrane and cytoplasmic regions, with an inner positively charged polypeptide segment, a membrane spanning hydrophobic region, and an outer negatively charged polypeptide region. Each contains a conserved tyrosine residue in the cytoplasmic sequence (potentially a phosphory- lation site), and a conserved cysteine residue in the membrane spanning region (potentially a palmitoylation site). Virtually all cysteine residue positions are conserved among species. Together with the conservation of the N-glycosylation sites and the conservation of amino acid residues throughout the polypeptide, this suggests tha t the overall s t ructure of lactase in different species is very similar (Fig. 1).

During insertion of lactase into the apical microvillus membrane, it is proteolytically processed, resulting In a mature membrane bound enzyme and an additional peptide (Fig. 1). The membrane-bound part, comprising the C-terminal two regions (region III and IV), comprises the functional intestinal enzyme lactase. The fate of the released peptide (regions I and II), a f ter processing, is controversial (8,9,10). Data in ra t indicate tha t the N-terminal regions (I and II) are proteolyt ical ly released into the lumen (11).

LACTASE GENE EXPRESSION

CLINICAL RELEVANCE

777

Lactase phenotypes

Lac tase plays a c r i t i ca l role in the nutr i t ion of human and o ther mammal ian neonates, s ince this enzyme is the only smal l in tes t ina l brush border hydrolase responsible for the digest ion of lac tose . During deve lopment in v i r tua l ly all mammals , l ac t a se spec i f ic ac t iv i ty is high around the t ime of b i r th and decl ines in the weaning period. Around weaning, a t rans i t ion occurs in the expression of microvil lus membrane hydrolases. This is cha rac t e r i zed by an increase in suc rase - i somal ta se and alkal ine phosphatase ac t iv i t ies . Sucrase - i somal tase increases during fur ther deve lopment and remains a t high levels throughout l i fe (12,13). This t rans i t ion of enzymat i c ac t iv i t i es enables developing mammals to switch f rom milk to a non- milk diet .

Lac tase is not regu la ted by the presence of i ts subs t ra te (14); adminis t ra t ion of lac tose does not e l eva t e l ac t a se spec i f ic ac t iv i ty . In adul t rabbi ts and ra t s no adapta t ion of l ac tase ac t iv i ty is observed in the presence of milk or lac tose in the die t (15). However, i t has been repor ted tha t , i r r espec t ive of i ts source, a high concent ra t ion of glucose s l ight ly increases lac tase ac t iv i ty in r a t (16). The independence of l ac tase development from subs t ra te supply is emphasized by the f ac t t ha t l ac tase gene expression is increased prior to bir th, and high levels of ac t iv i ty a re presen t a t bir th (17). Thus, all evidence suggests t ha t the level of l ac tase ac t iv i ty is genet ica l ly determined; only minor control may be exe r t ed by glucose, but no regula t ion is exe r t ed by lactose . In humans nei ther prolonged ingest ion of lac tose nor e l iminat ion of l ac tose from the d ie t a l t e r reduced lac tase ac t iv i ty (18,19).

In the ma jo r i ty of the human population, the pa t t e rn of l ac tase ac t iv i ty during development is cha rac t e r i zed by an increase during la te fe ta l per iod to high levels around birth. A decrease of ac t iv i ty occurs at around age 5 years , leading to low ac t iv i ty levels in adulthood. A minor i ty of the human population, however, re ta ins high levels of ac t iv i ty throughout adul t l i fe (20,21). Highest levels of ac t iv i ty a re always found in the proximal jejunum, although absolute l ac t a se ac t iv i ty levels a re higher in the l ac t a se -pe r s i s t en t phenotype (22).

In adul t humans of Northern European ex t rac t ion and of a few other groups, the abi l i ty to digest milk is considered normal. However, for most humans and probably all o ther adul t mammals , s ignif icant milk ingestion resul ts in mi ld to severe gas t ro in tes t ina l complaints , caused by low lac tase levels and the inabil i ty to digest lac tose (23). This has been t e rmed late onset lactase deficiency. Unfortunate ly , the t e rm lactase deficiency has become establ ished in the l i t e ra ture , even though i t is now c lear tha t low lac tase ac t iv i ty is not a def iciency, but a condit ion found normal ly in most adult humans, as well as all o ther adul t mammals tha t have been s tudied in this respect . This te rminology is derived f rom ini t ial s tudies per formed in adult human subjects of Northern European origin, whom we now know mainta in high lac tase levels beyond infancy. These init ial f indings led to the assumption tha t high l ac tase ac t iv i ty throughout l i fe was normal. Subsequent observations of a lmost to ta l absence of l ac tase ac t iv i ty in small in tes t inal biopsies of heal thy adults in o ther r ac ia l and ethnic groups led to the designat ion of such people as lactase deficient (24,25,26). Extensive population studies showed tha t l ac tase levels a re low in the major i ty of the world 's adul t population with a cha rac t e r i s t i c rac ia l dis tr ibut ion (20). Never theless , the te rminology s t i l l implies tha t low lac tase levels in adul t humans represen t an abnormali ty .

Populat ion gene t ic analysis has indica ted tha t e leva ted l ac ta se ac t iv i ty in adul t l i fe is inheri ted as a single autosomal dominant gene (20). However, a genera l ly accep ted mechanism of regula t ion to explain the various phenotypes of l ac tase ac t iv i ty within the world 's


populat ion has not y e t been descr ibed, and is the focus of many r e c e n t invest igat ions. Studies showing tha t l ac tase mRNA in humans, ra t s , and rabbi ts changes coord ina te ly with the amount of enzyme ac t iv i ty suggest t ranscr ip t iona l control of l ac tase expression (7,21,27,28,29,30,31). Deta i led informat ion on the regula t ion of l ac t a se expression will be p resen ted below.

Lactose intolerance

The inabi l i ty to digest lac tose as a resul t of low lac tase levels may lead to abdominal complaints . Typical symptoms induced by the ingestion of milk and/or milk products containing lac tose include abdominal pain, cramps or dis tent ion, nausea, f la tu lence and diarrhea; in chi ldren and adolescents , vomit ing may occur. A va r i e ty of t e rms has been used to descr ibe cl inical symptoms re l a t ed to the ingestion of milk and/or the absence of lac tase . Lactose intolerance is cha rac t e r i zed by occurence of these symptoms a f t e r the ingestion of lactose . The t e rm lactose malabsorption is reserved for those pa t ien ts in whom impaired in tes t inal uptake of lac tose has been proven by an appropr ia te t e s t (32). Lactase deficiency is def ined by low (>2 SD below the mean), or very ra re ly no l ac ta se ac t iv i ty in a small in tes t inal biopsy appropr ia te ly assayed for enzymat i c ac t iv i ty (24,33,34). Normal values have been descr ibed for children, and for adults f rom d i f fe ren t rac ia l background (35). Lac tase def ic iency is e i ther a p r imary or secondary event . Primary lactase deficiency occurs in p r ema tu re infants as a resul t of immature in tes t inal development , or as an ex t r eme ly ra re cl inical syndrome (34,36). This l a t t e r syndrome, ca l led human congeni ta l l ac t a se def ic iency, is inher i ted in an autosomal recess ive fashion, resul t ing in the absence of ac t ive lac tase (34,37). The most common form of decreased l ac tase ac t iv i ty of pr imary cause appears in the major i ty of the worldts populat ion around the age of 5 years; t h e r e a f t e r only low lac tase levels pe rs i s t (see sect ion lactase phenotypes). Secondary lactase deficiency is caused by in tes t ina l mucosal injury (36). Diseases tha t can cause mucosal damage or villus f la t tening include infect ious gas t roen te r i t i s , pa ras i t i c infect ions, ce l iac disease, t ropica l sprue, radia t ion en te r i t i s , drug-induced enter i t i s , and Crohn disease involving the small in tes t ine (38,39).

Among people lacking substant ia l levels of l ac tase ac t iv i ty , symptoms of malabsorpt ion may s t i l l va ry as a resul t of more or less adequa te adapta t ion of the in tes t ina l flora. Feca l f lora a re known to adapt to the types and amounts of ingested carbohydra te , and i t appears tha t quant i t ies of lac tose , which are not hydrolyzed in the smal l in tes t ine , can be salvaged by the colonic f lora by fe rmenta t ion to shor t -chain f a t t y acids, hydrogen, methane , water , carbon dioxide and the production of energy (40). If lac tose is provided in smal l amounts over a longer per iod of t ime in lactose intolerant people, the flora may adapt to the lactose- load, and consequent ly the symptoms produced by gas and acid in the colon may be reduced or e l imina ted (41). This mechanism of lac tose to le rance probably accounts for the discrepancy in some studies in which people with low l ac ta se levels were not considered lac tose in to lerant (42). Dif ferences in colonic distension and in individual responses to pain may also vary among lac tose in to le ran t people, providing o ther var iables which account for the sever i ty of symptoms a f t e r l ac tose ingestion.

The diagnosis of l ac tose malabsorpt ion is based on a combinat ion of c l inical findings and the resul ts of appropr ia te t es t s (32,43,44,45,46). The assay of l ac tase ac t iv i ty in small bowel biopsies es tabl ishes the amount of funct ional l ac tase available; normal values have been published (35). However, when lac tase def ic iency is caused by in tes t ina l injury, the lesions may be focal or patchy. Consequently, in tes t ina l biopsies may not show abnormal l ac t a se levels (42). In this respec t , i t is of in te res t t ha t a pa tchy pa t t e rn of l ac t a se pro te in expression is found in infants during chronic d iar rhea (47). Thus, the locat ion of sampling of in tes t inal biopsies de te rmines the outcome of the assay. I t should also be noted tha t the t e s t is invasive and t ime consuming. In the past , conf i rmat ion of lac tose malabsorpt ion is accomplished using

LACTASE GENE EXPRESSION 779

the lactose absorption tes t (32), which measures the uptake of monosaccharides after lactose hydrolysis. This tes t has a sensitivity and specificity of 75% and 96%, respectively. However, in children as well as in adults, it is cumbersome, invasive, and t ime consuming, and has largely been replaced by the lactose breath hydrogen tes t (46,48). Although the la t ter test really measures lactose non-absorption rather than lactose hydrolysis and monosaccharide uptake, it is highly sensitive and specific (32). In both children and adults this test is superior to the lactose absorption test , and it is simple and non-invasive (49). Treatment of lactose intolerance is accomplished by reduction or restriction of dietary lactose intake. Alternative nutrient sources to avoid reductions in energy and protein intake should then be provided, and adequate calcium intake should be guaranteed. Commercially available enzyme substitutes to digest lactose in dairy products can be of additional benefit (38,39).

Human

Rabbit

I II III IV I I I I I I I I

1 YY Yyee I

I I I I I I I ( ~ I I I I I I I I I I CC CCCC C CC C C C C CCCC

1 ee I

I I I I I I I I I I I I I I I I I CC CCCC C CC C C C C CCCC

I WYY e �9 Rat ]

I ]] ]I II ] I ] II ] c c c c c c c c c c c c c

i , , , , I | I

AA 0 500 i 000 1500 I

2000

FIG. 1. Comparison of amino acid sequences from human, rabbit and ra t lactase. Darkly shaded areas represent highly homologous sequences between the respective regions (I-IV). Lightly shaded areas represent sequences with low homology between the respective regions. The white box indicates the amino-terminal signal sequence. The black box indicates the carboxy-terminal transmembrane sequence. Y indicates a potential N-glycosylation site. C indicates a cysteine residue. E indicates the glutamate residue, which represents the cri t ical residue in each of the two mapped active sites of lactase at position 1271 and 1747, respectively. Arrow indicates the proteolytic processing si te (Data from references 6,112,118).


MOLECULAR AND CELLULAR ASPECTS OF LACTASE

The lactase gene and promotor

The gene for human lactase, located on chromosome 2 (50), is comprised of 17 exons and covers approximately 55 kb (51). Transcription and splicing give rise to an mRNA of slightly more than 6 kb in humans and rabbits (6), and rats (52). Detailed comparison of the mRNA from humans with lactase persistence or hypolactasia demonstrated that they were virtually indistinguishable, with the exception of a few point mutations which did not affect the amino acid structure (51). The difference in lactase expression could therefore not be a t t r ibu ted to d i f fe rences in the s t ruc ture of the gene i tself , cons is ten t with the hypothesis tha t d i f fe ren t ia l gene regula t ion is responsible for the two phenotypes. How lac tase gene expression might be contro l led is beginning to be examined by analysis of the 5' flanking region of the human, ra t , and pig lac tase genes. One kb of the human 5' f lanking region was sequenced and examined for consensus binding s i tes for known t ranscr ip t ion fac tors (51). Pu ta t ive binding s i tes for a number of common factors , including Spl , SRF, AP2, and CTF/NF-1 , have been ident i f ied. Although there is evidence tha t g lucocor t icoids regula te human lac tase levels, no g lucocor t icoid response e l emen t was ident i f ied in this region. The 1 kb sequence of the r a t 5' flanking region has been compared to tha t of the human gene, and i t was found tha t the f i rs t 155 bases from the t ranscr ip t ional s t a r t s i t e showed 72% s imi lar i ty , while the more d is tan t sequence decreased to 50% s imi la r i ty (53). In addit ion, potent ia l CTF/NF and AP2 s i tes have been found in both human and ra t , while CREB, O c t l / O c t 2 , SRF, and SP1 s i tes , which were ident i f ied in the human sequence, were not p resen t in the r a t (53). On the o ther hand, the r a t showed potent ia l binding s i tes for a ca lc ium response e l emen t and C/EBP, not p resen t in the human sequence. Ra t l ac tase levels a re c lear ly regu la ted by both cor t ico id and thyroid hormones, but ne i ther binding s i t e is ident i f iable within the f i rs t 1.2 kb of the r a t f lanking region. Unfortunately, there a re as y e t no functional da t a avai lable to de te rmine which, if any, of these pu ta t ive regula tory s i tes a re ac tua l ly involved in lac tase control. Regula t ion of pig lac tase was analyzed using a d i f fe ren t approach (54). By footpr int ing of the pig l ac tase flanking region, a region from -40 to -54, which bound a nuclear pro te in (NF-LPH1) tha t var ied coordina te ly with l ac t a se ac t iv i ty , was ident i f ied. The same sequence was ident i f ied in the human lac tase gene and appears to be funct ional in the human intes t inal cel l l ine Caco-2 (55). These da ta suggest tha t this f ac to r may be involved in the decl ine in l ac t a se expression.

Lactase gene transcription

Fur the r evidence tha t l ac tase levels a re pr imar i ly regu la ted a t the level of gene t ranscr ip t ion come from studies cor re la t ing the level of mRNA with tha t of protein. Although ear ly repor ts indica ted no cor re la t ion be tween the two (56,57,58), more r e c e n t de ta i led studies indicate a c lose corre la t ion . Comparison of to ta l r a t l ac tase ac t iv i ty and to t a l l ac tase mRNA along the in tes t ine and during development indicated a coord ina te change in both (52). When lac tase enzyme ac t iv i ty and l ac tase mRNA in human pa t ien ts were compared in two d i f fe ren t studies, l ac t a se enzyme levels were shown to be closely c o r r e l a t e d with mRNA (27,28). A careful analysis of l ac t a se expression during development and along the in tes t ine , in which l ac tase enzyme ac t iv i ty assays, quant i ta t ion of l ac tase pro te in by immunoelectrophoresis , and quant i ta t ion of l ac tase mRNA by r ibonuclease pro tec t ion assay were ca r r i ed out, c lear ly


demons t ra ted t ha t all t h ree changed in concer t . In addit ion, measurements of l ac tase t ranscr ip t ion levels co r re l a t ed with the pa t t e rn of lac tase enzyme (30). Thus, while addit ional regu la tory mechanisms have been indicated by other studies and may have a role in de termining the final amount of enzyme (56,57,58,59,60), the major regula t ion of l ac tase enzyme levels is a t the level of t ranscr ip t ion of the lac tase gene.

Studies of t ransp lan ted r a t fe ta l in tes t ine have shown tha t the decrease in lac tase ac t iv i ty is de te rmined by the age of the t issue, not of the host (61). Thus, the underlying pa t t e rn appears to be imprinted in the t issue, and does not depend on hormonal changes. Many studies, using in tes t inal isograf ts , have cont r ibuted to the concept t ha t pos i t ion-appropr ia te d i f fe ren t ia t ion along the proximal to dis tal axis of the smal l in tes t ine can occur in the absence of exposure to luminal contents , and the re fo re conclude tha t such regula t ion in the small in tes t ine is genet ic (62).

Studies in r a t indicate t ha t thyroxine is a modula tor of the developmenta l pa t t e rn of l ac tase expression a t the t ime of weaning. Hypophysectomy and thyro idec tomy re t a rd the decrease in l ac t a se ac t iv i ty during the third pos tna ta l week, while thyroxine r ep lacemen t res tores the normal pa t t e rn (63). Glucocor t icoids enhance r a t l ac t a se ac t iv i ty in the f i rs t weeks of l i fe (64), and increase lac tase mRNA conten t a t 6 days of age (65). Recen t studies in r a t demons t ra ted coopera t ive e f fec t s of thyroxine and g lucocor t ico id hormones in modulat ing the pos tna ta l deve lopment of l ac tase (65,66). An increase in l ac tase speci f ic ac t iv i ty following inject ion of ep ide rmal growth fac tor (EGF) has been observed (67,68).

There are few da ta avai lable on hormonal regulat ion of l ac t a se in human intest ine. Experiments with explaots of 12-14 week ges ta t ion human intes t ine showed tha t hydrocort isone induced an increase in lac tase levels (69). However, the physiological re levance of this finding is not c lear , as the major increase in human l ac t a se occurs in the las t few weeks of fe ta l l ife. A la te ges ta t ional upsurge in fe ta l serum cor t i sol levels has been descr ibed in humans (70), but whether this has a regu la tory role is cur ren t ly unknown. Moreover, a consensus binding s i te for g lucocor t icoid r ecep to r was not found in the r epor ted p romoter region of l ac tase {51).

Translational and post-translational processing of lactase

From ini t ia t ion codon to s top codon, human lac tase mRNA encodes 1927 amino acids forming the comple te t rans la t ion product . Deta i l s of i ts processing have largely been deduced from e i ther i ts p r imary sequence or studies of its biosynthesis. The mass of the nascent polypept ide is approximate ly 190 kDa. A signal sequence of about 3 kDa is presumably co- t rans la t ional ly c leaved off the polypeptide. Membrane t rans loca t ion proceeds up to a hydrophobic amino acid sequence a t positions 1883 to 1901, which functions as t rans loca t ion stop, yielding a type I t r ansmembrane prote in with only a small cy top lasmic tai l . Studies in mammals , including human, pig, rabbi t , and ra t , have shown tha t the ini t ial in t race l lu la r form of l ac tase is 200-220 kDa (8,71,72,73). Biosynthet ic incorporat ion of [3H]mannnse into the l ac tase precursor , and its endoglycosidase-H sens i t iv i ty indica ted tha t this form contains high- manonse N-linked ol igosacchar ides (71,72,73,74). These high-mannnse N-linked ol igosaccharides a re then modif ied, eventual ly leading to the format ion of complex N-linked ol ignsaccharides during the t r anspor t of the l ac tase precursor f rom the endoplasmic re t icu lum to the t rans-Golgi complex. In addition, some O-linked glycosylat ion of the l ac tase precursor takes p lace in the Golgi complex (75,76). Together , both modif ica t ions yield a complex glycnsyla ted lac tase precursor of sl ightly higher apparent molecular mass (approximate ly 220- 240 kDa) than the ini t ial high-mannose precursor .

As ident i f ied with immunohistochemical techniques (17,77,78), l ac tase prote in is predominant ly presen t in the apical , microvil lus membrane of smal l in tes t inal en te rocy tes at


i ts funct ional s i te . Many o ther in tes t inal prote ins have also been found to res ide pr imar i ly in the microvil lus membrane: e.g. aminopept idase , sucrase- i somal tase , and alkal ine phosphatase (9,79). Informat ion regarding the mechanism(s) responsible for this po lar ized local iza t ion of proteins is very l imited. General mechanisms for this polar iza t ion could include: sort ing of de novo synthesized prote ins by means of vec to r ia l ves icular t ranspor t , r e l a t i ve increased turnover of prote ins a t one membrane domain, or d i f fe ren t ia l e f f ic iency of t ranscytos is from the baso la te ra l p lasma membrane to the apical p lasma membrane. Each of these mechanisms is capable of genera t ing polar ized dis t r ibut ion of individual prote ins in ce r t a in epi the l ia l cel l types (80,81,82). Thus far , the speci f ic mechanisms control l ing l ac tase sor t ing in normal en te rocy tes have remained elusive. However, s tudies of e n t e roc y t e membrane prepara t ions have never found prote ins with the molecular mass of l ac tase on the baso la te ra l membrane, suggesting tha t de novo synthesized lac tase is very e f f i c ien t ly sor ted to the apical membrane.

Messenger RNA ta rge t ing may provide an a l t e rna t ive mechanism for in t race l lu la r local iza t ion of proteins. For example , mRNA sort ing seems to con t r ibu te to polar ized expression of actin, s ince the highest in t race l lu la r levels of ac t in mRNA are presen t a t the apical side of mouse en te rocy tes as found by in si tu hybridizat ion (83). Co- loca l iza t ion of act in mRNA and act in pro te in has been observed (83). Fur thermore , in situ hybridizat ion has shown tha t mRNA sort ing can be demons t ra ted for many o ther mRNAs in polar ized cells, e.g. f ibroblasts , Xenopus oocytes , neurones and Drosophila embryos (84,85,86,87). Also for lactase , mRNA sort ing seems to cont r ibu te to polar ized expression, s ince highest in t race l lu la r levels of l ac tase mRNA are present in the apical domain of ra t , rabbi t , and human en te rocy tes , as found by in situ hybridizat ion (77,88).

A f t e r comple te glycosylat ion and t ranspor t , specif ic c leavage of the precursor genera tes the ma tu re l ac tase prote in comprising regions III and IV (Fig. 1)(8). The local izat ion, mechanism, and function of this c leavage are s t i l l a m a t t e r of cont roversy (8,10,11). The function and local iza t ion of the c leaved N- te rmina l pept ide, compris ing amino acids 1 to 868 (region I and II), is not known. In the ra t , the 220 kDa complex g lycosyla ted precursor is p ro teo ly t i ca l ly c leaved on the microvil lus membrane in two s e p a r a t e steps: f i rs t , from 220 kDa to 180 kDa (a t rans ien t in te rmedia te observed by metabo l ic labeling) and then from 180 kDa to the final 130 kDa form (8). Similar processing with an in t e rmed ia t e s tep has also been ident if ied in rabbi ts (72). A comparable analysis in humans did not ident i fy an in te rmedia te step, but showed d i r ec t processing from the 240 kDa complex g lycosyla ted precursor to the 160 kDa ma tu re form (71,73). Nevertheless , p ro teo ly t i c processing of human l ac ta se is c lear ly a post-Golgi event , occuring e i ther in the trans Golgi network, t r anspor t ves ic le or a f t e r insert ion into the apical membrane (10,89). In r a t intes t inal explant studies, the uncleaved enzyme is inser ted into the microvil lus membrane as a complex g lycnsyla ted precursor of approximate ly 220 kDa, indicat ing tha t proteolysis of the precursor takes p lace a f t e r arival on the apical membrane (8). However, in studies with human biopsies as well as with the human Caco-2 cel l lines, the re are indications of in t race l lu lar processing of the lac tase precursor , but these studies do not exclude cel l surface-process ing (71,73,90,91).

The locat ion of the p ro tease responsible for l ac tase processing is s t i l l controversial . The p ro teo ly t i c processing of l ac tase in in tes t inal explants of r a t and rabbi t occurs in the absence of luminal (pancreat ic) proteases , and there fore may proceed through integral membrane protease(s) (8,72). In contras t , the c leavage of sucrase - i somal tase has been shown to depend on panc rea t i c pro teases (61,90), and studies in in tes t inal segments of in tac t ra t s suggest t ha t the final cel l sur face processing of l ac tase occurs by luminal pro teases (11). Interest ingly, a processed ma tu re lac tase pro te in has also been isola ted from microvil lus membranes of Caco-2 cel ls (9).

Studies have been d i rec ted at whether the p ro teo ly t i c processing of l ac tase is essent ial for e i ther in t race l lu la r t a rge t ing or enzymat ic ac t iv i ty of the enzyme. Transfect ion of COS


cel ls with a full length human l ac ta se cDNA resul ted in the synthesis and t ranspor t to the p lasma membrane of a complex glycosyla ted , high molecular weight form of lac tase , with full enzymat i c ac t iv i ty (10). Thus, c leavage is apparent ly not essent ia l for e i ther cel l sur face expression or enzymat i c ac t iv i ty . The role of p ro teo ly t i c processing in in t race l lu la r t r anspor t of human lac tase was fur ther s tudied in polar ized, non- intes t inal Madin Darby canine kidney (MDCK) cel ls (92,93). A f t e r t r ans fec t ion of a full length human l ac ta se cDNA into these cells , l ac tase was inser ted into the apical and baso la te ra l membrane: 72% and 28% respect ive ly . Of the lac tase present in the apical membrane, 42% was in the ma tu re 160 kDa form. Of tha t s o r t e d baso la te ra l ly , 20% was in the 160 kDa form. These resul ts ind ica te tha t p ro teo ly t i c processing occurs a f t e r sort ing and is not necessary for sur face expression.

Many intes t inal apical microvil lus membrane prote ins are thought to be homodimers (94), and there are indicat ions tha t d imer iza t ion may be a regu la tory event in in t race l lu la r t ranspor t . Lac tase has been shown to d imer ize l a t e in in t race l lu la r t ranspor t , suggesting tha t this is a t rans-Golgi or cel l sur face phenomenon (94). The re la t ionship be tween d imer iza t ion and pro teo ly t ic processing of l ac tase remain to be e lucidated .

Several polymorphisms regarding glycosylat ion of l ac tase have been described. F i rs t , a shif t in N-linked glycan te rmina l sugar composit ion from s ia l ic acid to fucose occurs a t the t ime of weaning, as shown in ra ts (75). This change, however, most l ikely only r e f l ec t s changes of the s ia lyl- and fucosyl -g lycosyl t ransferase ac t iv i t ies in the en t e rocy te s during weaning (95). This a f fec t s the dis tr ibut ion of t e rmina l monosacchar ides on o l igesacchar ides of many o ther microvil lus membrane proteins around weaning (96). This change in g lycosyla t ion does not account for the change in spec i f ic enzyme ac t iv i ty descr ibed for l ac t a se around the t ime of weaning (75). He te rogene i ty in O-glycosylat ion is found in l ac t a se immunoprec ip i ta ted from the brush border of human in tes t ina l biopsies. Approx imate ly 50% of all immunoprec ip i ta ted ma tu re l ac tase contained only N-linked glycans, while the o ther 50% conta ined both N- and O-l inked glycans. The presence of O-l inked glycans has been shown to e f f e c t enzyme ac t iv i ty , s ince absence of O-glycosyla t ion resul ts in a fourfold drop of the Vmax, whereas both forms have a s imilar K m (76). Nei ther the origin of this he te rogene i ty nor i ts biological re levance a re ye t known.

Localization of lactase expression

In our studies regarding the expression of l ac t a se in r a t during development , i t appeared tha t l ac tase mRNA was f i rs t de t ec t ab l e in the proximal r a t in tes t ine a t 18 days of ges ta t ion (17). At this s tage, a single layer of cuboidal cel ls is formed out of the und i f fe ren t i a ted s t r a t i f i ed epi thel ium, which co r re l a t e s with the appearance of l ac tase mRNA. The r a t l ac tase prote in was f i rs t ident if ied a t 20 days of ges ta t ion , just prior to birth. A t this t ime both l ac ta se mRNA, as well as protein, was presen t in all en te rocy tes along the villus. No lac tase mRNA or pro te in was ever d e t e c t e d in the intervi l lus region. Almost immedia te ly a f t e r bir th, l ac tase mRNA was r e s t r i c t ed to the lower half of the villus, whereas l ac tase pro te in was s t i l l p resen t over the whole length of the villus. This pa t t e rn of expression is main ta ined during fur ther deve lopment into adulthood. Interes t ingly , a s imi lar pa t t e rn of gene expression along the crypt -v i l lus (vert ical) axis of the small in tes t ine is found for r a t and human sucrase - i somal tase mRNA and protein (97,98).

Along the the proximal to d is ta l (horizontal) axis of the small in tes t ine , l ac tase ac t iv i ty is p resen t in a cha rac t e r i s t i c gradient in humans and o ther mammals (22). During ear ly pos tna ta l deve lopment of ra t , l ac t a se is present along the to ta l length of the small intes t ine, as well as in the proximal colon (99). The changes of l ac t a se ac t iv i ty along the horizontal axis of the r a t small in tes t ine around weaning have been the subject of our r ecen t invest igat ions, and have been compared to the regula t ion of sucrase - i somal tase a t the ce l lu lar and molecular


level (30,100). F rom 0-16 days of pos tna ta l development , l ac tase mRNA and pro te in were abundant along the to ta l length of the small in tes t ine . However, a t 21 days, l ac t a se mRNA and pro te in were no longer d e t e c t a b l e in the te rmina l ileum. F rom 28 days on, zones of reduced l ac tase expression were found in the duodenum and ileum. These zones displayed pa tchy expression of l ac tase pro te in among en te rocy te s along the villus (I 00, I0 I). In cont ras t , the expression of sucrase - i somal tase was f i rs t d e t e c t e d at 16 days; pa tchy expression of sucrase - i somal tase pro te in was found along the en t i r e length of the small in tes t ine (I00). At 21 days, expression of sucrase - i somal tase was abundant along the t o t a l length of the small intest ine. These concordant changes in both l ac t a se mRNA and pro te in during development suggest t ha t the horizontal gradient of l ac tase expression is es tabl ished by t ranscr ip t ional regula t ion (30,100). Although lac tase and sucrase - i somal tase are d i f f e ren t ly regu la ted along the horizontal axis, the comparable pa t t e rn of expression of the r e spec t ive mRNAs and prote ins of these d isacchar idases along the crypt -v i l lus axis suggests tha t they may share some common regula tory mechanisms for expression along the ve r t i ca l axis. The zones of pa tchy lac tase expression descr ibed above appear around weaning and f lank the a rea of high lac tase expression in the mid- in tes t ine . Patchy expression is not found exclusively for lac tase , s ince pa tchy expression is also found for sucrase - i somal tase before weaning in r a t small in tes t ine (I00), and for blood group A antigen in humans (102).

Regulat ion of the pa tchy expression of l ac t a se protein is d i f f icu l t to explain. This pa t t e rn exists among villus en te rocy tes of which only a var iable number along the villus randomly express lac tase . The two types of en te rocy tes have not been dist inguished morphological ly or otherwise. At the ext reme, l ac t a se prote in is occasional ly found in only about 1% of the villus en te rocy tes in the most per iphera l zones of the horizontal axis of l ac tase expression (I00). Star t ing from these per iphera l zones of expression towards the cen te r of the smal l intest ine, the pe rcen tage of l ac tase -pos i t ive cel ls rapidly increases to I00%. It is not c lea r what this pa tchy l ac tase expression ac tua l ly represents . Possible mechanisms of control may ac t a t the t ranscr ip t ional , t rans la t ional , or pos t - t rans la t iona l levels. In rabbi t , this pa t t e rn has also been demons t ra ted in a s tudy in which lac tase ac t iv i ty and pro te in were analyzed along the length of the in tes t ine in animals of I, 7, 15, 30 days and 3-month-old adults. The development of l ac tase speci f ic enzymat i c ac t iv i ty was characterized by re la t ive high levels unti l weaning (28 days in rabbit) and a r e l a t ive ly rapid fal l of speci f ic ac t iv i ty soon t he r ea f t e r to 5-10% of the preweaning levels. In preweaning animals, all en te rocy tes along the small in tes t ine demons t ra te lac tase immunoreac t iv i ty . In adul t rabbi t , uniform immunoreac t iv i ty for l ac tase is only seen in the middle par t of the smal l in tes t ine whereas a pa tchy pa t t e rn is seen in proximal and distal regions (101). Pa tchy pa t t e rn s in these animals resemble l ac tase pa t t e rns as found in human biopsies (78,103).

A fur ther example of pa tchy gene expression in en te rocy tes was r ecen t ly found in the expression of blood group A antigen. The responsible r was d is t r ibuted in a pa tchy fashion along human intest ine of blood group A non-secre tors (I02). The phenomenon was not the resul t of d i f ferences in the ac t iv i ty levels of r ga lac tosaminot ransfe rase , which is also necessary for biosynthesis of the A antigen. I t is unlikely tha t expression is co r r e l a t ed with s tem cel l l ineage, s ince no sheets or s t r e tches of posi t ive cel ls were found, but instead, random pa tches were ident i f ied along the length of the villus. Finally, the re was no cor re la t ion with sucrase - i somal tase gene expression, which was uniformly dis t r ibuted. We suggest tha t patchiness r e f l ec t s very subt le d i f ferences in ce l lu lar d i f fe ren t i a t ion s t a t e . Pa tchy pa t te rns seem to r e f l e c t a genuine mechanism of gene expression in the in tes t ine of man, rabbi t , and ra t , and most l ikely in many more species.


Enzyme specificities of lactase

Lactase isolated from microvillus membranes of enterocytes exhibits at least four characteristic enzyme activities: lactase (B-D-galactoside galactohydrolase, EC 3.2.1.23), phlorizin hydrolase (phlorizin glucohydrolase, EC 3.2.1.62), and 13-glucosyl- and B- galactosylceramidase (EC 3.2.1.45 and 46), respectively (104,105,106,107,108,109). The specificity of lactase towards a broad range of different B-glycosyl-compounds may contain important clues for the biological role(s) of the enzyme. Its role in lactose digestion in young mammals is evident, but digestion of other dietary components may be important as well. Lactase appears to have low specificity with respect to the aglycan part of the substrates, which gives rise to B-glycosidase activity towards a large number of naturally oceuring and artifical substrates. The strong affinity of this enzyme for glycosylceramides, in combination with the finding of appreciable amounts of active lactase enzyme in the adult rat intestine, suggest a possible role for lactase in adulthood in the digestion of glycolipids (104}. Another possible role may be found in the digestion of cellulose, the most important compound containing B(l,4)glucosidic bonds; pig lactase has been shown to exert activity towards cellulose and cellodextrins (110). It is, however, not known if this B-glucosidase activity of lactase contributes significantly to the digestion of the approximately 4 grams of cellulose ingested daily by humans.

Substrate specificity has been studied using a series of chemically defined substrates resembling lactose. It was shown that changes in the glucose part of the lactose molecule have a much greater inhibitory effect on hydrolysis than changes of the galactose part. All the hydroxyl positions in the molecule were changed independently into either hydrogen residues or methyl residues, and the glucose moiety, in particular the OH-6, proved to be essential (111).

The multiple enzyme specificities are found on the single polypeptide corresponding to the mature form of lactase, comprising regions Ill and IV (Fig. 1), which can be immunoprecipitated with a monoclonal antibody from rat, pig, lamb, and human small intestine (9,73,79,104,11 I). The enzymatic activities have been recently assigned to two active sites on the mature protein characterized as B(1,4)galactosidase and l~(1-4)glucosidase (112). These sites have been studied in rabbit and human using conduritol-B-epoxide (CBE) as an affinity label (112,113). CBE is a substrate analogue that binds covalently to carboxylated amino acids essential for the hydrolysis of glycosides. CBE has been used in the study of a variety of 13-glycosidases (114,115) as well as u-glucosidases (I 15,116). Inactivation studies of rabbit lactase with CBE strongly indicate that these activities originate from two different active sites. Glutamate (Glu) 1271 in region Ill was assigned to be part of the lactase (B-galactosidase) site, and glu 1747 in region IV was assigned to the phlorizin hydrolase (B-glucosidase) site (Fig. 1)(112).

Lactase belongs to a family of glycosidases in which glutamate is a critical active site residue. This is based on the alignment of these glutamate positions and their respective flanking regions with sequences of a large number of other B-glycosidases (5). In contrast with this family of B-glycosidases based on glutamate, another family of u-glycosidases, which includes sucrase-isomaltase, is characterized by the essential role of aspartic acid within the active site (I 16,117). The consensus sequence around the active site of B-glycosidases is X - glutamic acid - asparagine - glycine, where X can be threonine, serine, valine or methionine (112). Interestingly, no such consensus sequence is present in regions I and II of lactase, despite high homology between these regions and regions Ill and IV, which form the mature enzyme. This most likely indicates that no glycosidase activity resides in regions I or II (112).

Lactase has been shown to be a B-glucosidase, although for many years it was regarded a B-galactosidase. Evidence is based on sequence comparison, substrate specificities, and


mapping of the ac t ive s i tes (1,2,112). As a B-glucosidase, l ac tase may have mul t ip le functions beyond the per iod of lac ta t ion , s ince natura l subs t ra tes in the adul t d ie t (e.g. g lycosylceramides) can he hydrolyzed. In this respec t , the recogni t ion of l ac t a se as a genera l 6-glucosidase seems well just if ied.

SUMMARY

Lactase , a B-glycosidase with mul t ip le subs t ra te spec i f ic i t i es , is the key enzyme for the hydrolysis of the ca rbohydra te lac tose in mammal ian milk, and is of impor tance during the neonatal per iod of most mammals and humans. A f t e r the period of weaning, in the ma jo r i ty of the world 's populat ion and in v i r tua l ly all mammals , low lac tase ac t i v i t y is the norm. In cont ras t , high levels of ac t iv i ty are found in cer ta in human populations. Now it is c lea r t ha t the enzyme ac t i v i t y in these phenotypes is p r imar i ly regula ted a t the level of gene t ranscr ip t ion, as de l inea ted by studies in humans, as well as in o ther mammals . Fur thermore , as a resul t of dec reased t ranscr ip t ion of the gene, as observed in the phenotype cha rac t e r i zed by low adul t l ac t a se ac t iv i ty , a r e s t r i c t e d area of expression along the length of the smal l in tes t ine is found in adul t animals. Peak expression is found in the jejunum, whereas v i r tua l ly no expression is seen in both proximal and dis ta l ends of the intest ine. These areas with very low expression are cha rac t e r i zed by expression of l ac tase prote in on s ca t t e r ed en te rocy tes along the villus. A coord ina te decrease around weaning, as s tudied in pig, of both t ranscr ip t ion and of a nuclear f ac to r (NF-LPHI), which binds spec i f ica l ly to the l ac t a se promoter , may be an impor tan t molecu la r mechanism by which lac tase gene t ranscr ip t ion is regula ted. Fu tu re research will e luc ida te the role of this nuclear f ac to r in the d i f fe rences in l ac tase phenotypes among human populations.

ACKNOWLEDGEMENTS

We thank Chris Bor for preparing the figure. The work descr ibed f rom our labora tor ies was supported in pa r t by the Nether lands Organizat ion for Sc ient i f ic Research (NWO) (E.H.H.M.R.), by Nutr ic ia , Zoe te rmeer , and by the Irene Kinderziekenhuis Foundation, the Nether lands (H.A.B., E.H.V.B.); by NIH Research Grant # ROI DK 32658 and by the Cen te r for Gas t roen te ro logy Research on Absorpt ive and Sec re to ry Processes (NIH # P30 DK 34928) (R.K.M, R.J.G.), and by a NATO Col labora t ive Research Grant .

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Accepted for publication January 15, 1994.

lactase; origin, gene expression, localization, and function

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