Oligosaccharidases of the Small Intestinal Brush Border - ACS

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Oligosaccharidases of the Small Intestinal Brush Border GARY M. GRAY Division of Gastroenterology, Department of Medicine, Stanford University School of Medicine, Stanford, Calif. 94305

Carbohydrates represent an important and inexpensive source of calories for man but must be digested to monosaccharides before absorption can occur in the small intestine. Until the last few years, it was commonly believed that all hydrolysis occurred within the intestinal lumen under the influence of secretions from the intestinal wall, the so-called succus entericus. However, the work of Crane and his colleagues (1-3) localizing disaccharidase activities to the brush border membrane of the intestine drew attention to the potential role of the small Table 1 intestinal cell in carbohydrate diges DIGESTION OF CARBOHYDRATE tion. Table 1 out LUMINAL INTESTINAL lines important FOOD SOURCE %OFCHO HYDROLYSIS HYDROLYSIS carbohydrates in the diet of man, STARCH 60 —•MALTOSE, —•GLUCOSE the amounts and (AMYLOPECTIN MALT0TRI0SE, ot-DEXTRINS proportion of each AMYLOSE) ingested (4), and the site of hydro L A C T O S E 10 NONE —GLUCOSE +GALACTOSE lysis. Notably only starch and glycogen are par SUCROSE —•GLUCOSE NONE 30 + FRUCTOSE tially hydrolyzed within the intestinal lumen. Hydrolysis of the residual oligo saccharide products of starch and of the disaccharides sucrose and lactose occurs under the influence of enzymes integral to the intestinal brush border membrane. Intraluminal Digestion of Polysaccharide Despite the common belief of 15-20 years ago that starches are hydrolyzed completely to glucose, Whelan and his colleagues 05,6) carried out extensive experiments demonstrating that the final products under physiological conditions are the oligo saccharides maltose, maltotriose and the α-limit dextrins 181

Jeanes and Hodge; Physiological Effects of Food Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1975.


182

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

c o n t a i n i n g f i v e to nine glucose molecules and one or more α 1,6 branching l i n k s . This presumably r e f l e c t s a) the poor a f f i n i t y of α-amylase f o r α 1,6 branching l i n k s and f o r α 1,4 l i n k s a d j a cent to the 1,6 l i n k s (.5,6) and b) the preference of the a c t i v e s i t e of the enzyme f o r l i n e a r o l i g o s a c c h a r i d e s of f i v e or more glucose u n i t s by cleavage of the penultimate bond a t the reducing end of the molecule (7)· Despite claims that α-amylase secreted from the pancreas may b i n d to the i n t e s t i n a l s u r f a c e p r i o r to i t s a c t i o n on p o l y s a c charides ( 8 ) , i n t e s t i n a l f l u i d contains α-amylase at 10 times the c o n c e n t r a t i o n r e q u i r e d to e x p l a i n the i n v i v o s t a r c h h y d r o l y s i s i n man (9). Hence i t appears that d i g e s t i o n of s t a r c h and g l y cogen to o l i g o s a c c h a r i d e s i s p r i m a r i l y an i n t r a l u m i n a l process. Oligosaccharidases

of the I n t e s t i n a l

Surface

There i s only a t r a c e of o l i g o s a c c h a r i d a s e a c t i v i t y i n the l u m i n a l f l u i d s of the s m a l l i n t e s t i n e ; i n s t e a d , the carbohydrases are concentrated i n the brush border s u r f a c e membrane of the i n t e s t i n e . Table 2 l i s t s the enzymes from human s m a l l i n t e s t i n e that have been i d e n t i f i e d and c h a r a c t e r i z e d . A l l of these are l a r g e g l y c o p r o t e i n s . Notably there i s only a s i n g l e β-galactosidase (10) but s e v e r a l a-glucosidases (11,12) i n the brush border. There are other carbohydrases w i t h i n the i n t e r i o r of the i n t e s t i n a l c e l l that do not appear to have a d i g e s t i v e f u n c t i o n and these w i l l not be considered here. A l l of the a-glucosidases except t r e h a l a s e are capable of h y d r o l y z i n g maltose but i t seems p r e f e r a b l e to name them according to the s u b s t r a t e f o r which they are p e c u l i a r l y s p e c i f i c . The α 1,4 m a l t o - o l i g o s a c c h a r i d e and α-limit d e x t r i n products of amylase a c t i o n on s t a r c h are hydro l y z e d by glucoamylase and the α-dextrinase subunit of sucrase-ad e x t r i n a s e r e s p e c t i v e l y . The only i n t e s t i n a l carbohydrase that appears to have l i t t l e p h y s i o l o g i c a l r o l e i n d i g e s t i n g carbohy d r a t e i n the d i e t of modern man i s t r e h a l a s e s i n c e i t s appro p r i a t e s u b s t r a t e i s found only i n i n s e c t s and mushrooms. At l e a s t one of these enzymes, sucrase-a-dextrinase c o n s i s t s of a complex of two p r o t e i n s , each of which has i t s own, indepen d e n t l y a c t i n g enzyme s i t e (12). This p a r t i c u l a r h y b r i d enzyme has been cleaved i n t o a c t i v e , d i s t i n c t subunits of s l i g h t l y d i f f e r e n t molecular s i z e that r e t a i n the same biochemical c h a r a c t e r i s t i c s as found i n the n a t i v e h y b r i d (12). The α-dextrinase moiety i s commonly c a l l e d "isomaltase" because i t i s capable of h y d r o l y z i n g the 1,6 l i n k a g e s but the α 1,6 l i n k e d d i s a c c h a r i d e , isomaltose, i s not a saccharide product of amylase a c t i o n on s t a r c h and hence i s not a p h y s i o l o g i c a l s u b s t r a t e presented to the i n t e s t i n a l s u r f a c e . The reason f o r union of sucrase and α-dextrinase m o i e t i e s to form a h y b r i d molecule i s unknown s i n c e these enzymes, whether present i n h y b r i d or monomeric form, hydrolyze the a p p r o p r i a t e d i s a c c h a r i d e s u b s t r a t e i n an i d e n t i c a l


Jeanes and Hodge; Physiological Effects of Food Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1975. 2

9

*~ 1.1 (G )

4.3 ( i s o maltose)

20

3.8 (G )

18

280,000

210,000

280,000

Mol. Wt.

tG i n d i c a t e s glucose and the s u b s c r i p t the number o f α 1,4 l i n k e d glucose u n i t s .

*Commonly c a l l e d sucrase-isomaltase although isomaltose i s not a p h y s i o l o g i c a l s u b s t r a t e i n the i n t e s t i n e .

Trehalose

50

a-Dextrins

Trehalase

25

25

Sucrose

9

Sucrase-adextrinase*

2

Malto-oligosaccharides (G t >- G )

Lactose

Glucoamylase

a-Glucosidase

Lactase

β-Galactosidase

Enzyme Type Name

HUMAN BRUSH BORDER PLIGOSACCHARIDASES % Total Principal Maltase Substrate Activity Km mM

TABLE 2



184

PHYSIOLOGICAL

EFFECTS

OF

FOOD

CARBOHYDRATES

manner. However, experiments u s i n g a p u r i f i e d α-limit d e x t r i n as s u b s t r a t e have not yet been accomplished and i t i s p o s s i b l e t h a t the sucrase a c t i v e s i t e hydrolyzes adjacent α 1,4 l i n k a g e s w h i l e the α-dextrinase s i t e simultaneously hydrolyzes the α 1,6 branching p o i n t of the branched saccharide. Such c o o p e r a t i v i t y might g r e a t l y f a c i l i t a t e h y d r o l y s i s of the o l i g o s a c c h a r i d e to f r e e glucose. There i s some suggestion that l a c t a s e may a l s o e x i s t as a h y b r i d w i t h an a-glucosidase (13) and that glucoamyl a s e may be complexed w i t h an o l i g o 1,6 glucosidase (14) but s u f f i c i e n t l y pure preparations of these enzymes are not yet a v a i l a b l e to e s t a b l i s h t h i s . Development of Brush Border O l i g o s a c c h a r i d a s e s The human i n t e s t i n a l carbohydrases develop at v a r i o u s stages of u t e r i n e l i f e (15) as o u t l i n e d i n F i g u r e 1. The reason f o r the d i f f e r e n t i a l times f o r a c q u i s i t i o n of these enzymes i s unknown. Small i n t e s t i n a l c e l l s have a remarkably short l i f e span that appears to be r e g u l a t e d by the r a p i d maturation and m i g r a t i o n of c e l l s from c r y p t up along the v i l l u s f o r discharge of senescent c e l l s from the v i l l u s t i p . The o l i g o s a c c h a r i d a s e s , although not present i n the immature c e l l s of the i n t e s t i n a l c r y p t s , are acquired as c r y p t c e l l s develop m o r p h o l o g i c a l l y and migrate onto the v i l l u s , as shown s c h e m a t i c a l l y i n F i g u r e 2. R e g u l a t i o n of the Oligosaccharidases The feeding of sucrose (16), f r u c t o s e (16) or glucose (17) produces a doubling i n i n t e s t i n a l sucrase a c t i v i t y . Whether t h i s occurs by v i r t u e of an i n c r e a s e i n s y n t h e s i s of the enzyme or a decrease i n degradation, i . e . s t a b i l i z a t i o n , has not been c l e a r l y d e f i n e d but i t seems l i k e l y that feeding of carbohydrates r e t a r d s degradation of the enzyme (18,19). Although the synthe s i s and degradation of other o l i g o s a c c h a r i d a s e s have not been shown to be d i r e c t l y r e g u l a t e d by s u b s t r a t e or products, the mono saccharide products r e l e a s e d i n t o the i n t e s t i n a l lumen do compete f o r the a c t i v e h y d r o l y t i c s i t e , thereby r e t a r d i n g the r a t e s of h y d r o l y s i s (20). Role of Surface O l i g o s a c c h a r i d a s e s i n D i g e s t i o n The f i n a l o l i g o s a c c h a r i d e products from glycogen and s t a r c h d i g e s t i o n and the d i e t a r y d i s a c c h a r i d e s sucrose and l a c t o s e are hydrolyzed very e f f i c i e n t l y a t the brush border s u r f a c e of the i n t e s t i n e so t h a t the r e l e a s e d monosaccharides are produced i n great abundance. Hence, n e i t h e r h y d r o l y s i s i n the i n t e s t i n a l l u m i n a l contents by α-amylase nor s u r f a c e h y d r o l y s i s by o l i g o saccharidases i n t e g r a l to the i n t e s t i n e are r a t e - l i m i t i n g i n the o v e r a l l process of h y d r o l y s i s and t r a n s p o r t i n v i v o (21,22).


11.

185

Intestinal Oligosaccharidases

GRAY

LACTASE OTHER ouGLUCOSIDASES

TREHALASE


SUCRASE ISOMALTASE

FERTILIZATION

i

0

TERM

ι 4

ι 8

ι 12

ι 16

ι 20

ι 24

ι 28

ι 32

ι • 36

FETAL AGE (WEEKS) Figure 1. Development of human brush border oligosaccharidases in the fetus. The lines locate the range of fetal age during which full activity is acquired.

132 HRS

96 HRS

VILLUS

C R Y P T

ON A SYNTHESIS

PROTEIN DISACCHARIDASE SYNTHESIS ACTIVITY New England Journal of Medicine

Figure 2. Functional localization of intestinal cells from the time of their birth at the crypt base until cells migrate up the villus and are shed from the villus tip 132 hours later. Width of shaded vertical bars indicates relative amounts of the particular ac tivity, and vertical position denotes location of the activity in the crypt-villus unit.


186

PHYSIOLOGICAL

EFFECTS

O F FOOD

CARBOHYDRATES


Instead, r a t e - l i m i t i n g phenomena appear to be i n v o l v e d p r i n c i p a l l y i n the f i n a l t r a n s p o r t of the released monosaccharides. The s o l e exception to t h i s i s the h y d r o l y s i s of l a c t o s e which i s much slower than h y d r o l y s i s of other o l i g o s a c c h a r i d e s , as shown i n F i g u r e 3, so t h a t h y d r o l y s i s i s even slower than transport of r e l e a s e d monosaccharides (22). Thus normal man i s at a r e l a t i v e disadvantage f o r the d i g e s t i o n of l a c t o s e as compared to other d i e t a r y saccharides, and t h i s appears to become p a r t i c u l a r l y important when i n t e s t i n a l disease i s present. D e f i c i e n c y of I n t e s t i n a l

Oligosaccharidase

Since entry i n t o the i n t e r i o r of the i n t e s t i n a l c e l l i s reserved f o r monosaccharides, absence or marked r e d u c t i o n of an o l i g o s a c c h a r i d a s e r e s t r i c t s the o f f e n d i n g o l i g o s a c c h a r i d e to the i n t e s t i n a l lumen. This can have d i r e consequences, as o u t l i n e d i n F i g u r e 4 s i n c e the o l i g o s a c c h a r i d e a t t r a c t s water by v i r t u e of i t s osmotic f o r c e . As the saccharide passes down to d i s t a l small i n t e s t i n e and c o l o n , b a c t e r i a metabolize i t to two and three carbon fragments that are poorly absorbed i n lower bowel. Hence, the osmotic e f f e c t i s increased s e v e r a l f o l d so that i n g e s t i o n of only 50 grams of a d i s a c c h a r i d e may produce d i a r r h e a of 2000-3000 ml of f l u i d on an osmotic b a s i s alone. Other f a c t o r s such as the low pH produced by the metabolized fragments and s t i mulation of i n t e s t i n a l motion because of d i s t e n t i o n of the w a l l s of the hollow gut may a l s o c o n t r i b u t e to the d i a r r h e a and may s e c o n d a r i l y produce malabsorption of other n u t r i e n t s (Figure 4 ) . Primary Oligosaccharidase

Deficiencies

L a c t a s e D e f i c i e n c y . Lactase i s the i n t e s t i n a l saccharidase t h a t i s most commonly d e f i c i e n t , but the enzyme i s u s u a l l y normally a c t i v e i n c h i l d r e n and only becomes reduced i n a d o l e s cence and adulthood. I n t e r e s t i n g l y enough, most of the world's p o p u l a t i o n has a d u l t l a c t a s e d e f i c i e n c y (23-36), as shown i n Table 3. Thus, i t i s comTable 3 monly b e l i e v e d that l a c P R E V A L E N C E O F L A C T A S E DEFICIENCY tase d e f i c i e n c y i s a genGROUP % LACTASE DEFICIENT e t i c c o n d i t i o n . However, many of the r a c i a l groups WHITE w i t h a high prevalence of SCANDINAVIAN 3 adult lactase deficiency NORTH AMERICAN 5-20 s u f f e r from poor n u t r i BLACK t i o n and have an appre70 AMERICAN c i a b l e i n c i d e n c e of 50 AFRICAN OTHER small i n t e s t i n a l 80-100 disease^ c o n d i t i o n s CHINESE 55 INDIAN which are known to 95 FILIPINO depress i n t e s t i n a l 85 ABORIGINE (AUSTRALIAN) l a c t a s e out of ISRAELIS

60


11.

187

Intestinal Oligosaccharidases

GRAY

60 τ

or ι 50J


if)

ο

ι Q CO

40

30

20

10

Gastroenterology

Figure 3. Hydrolysis rates of lactose (L), maltose (M), and sucrose (S) from perfusion of a 30-cm segment of human jejunum in vivo (22). Brackets indicate ± 2 SE. Lactose is hydrolyzed much more slowly than the other disaccharides (P < 0.01). INTESTINAL L U M E N

SMALL INTESTINE

NO

LACTASE

LACTIC ACID

Annual Review of Medicine

WATERY DIARRHEA

MALABSORBTION FATS, PROTEINS, DRUGS

Figure 4. Schematic of the effect of disaccharidase defi ciency on the fate and action of a dietary disaccharide (see text for elaboration). If intes tinal lactase were present in normal concentrations, hydrol ysis would occur on the surface of the intestine and the mono saccharide products would be assimilated (41).


188

PHYSIOLOGICAL


p r o p o r t i o n to that found f o r other

EFFECTS OF

FOOD CARBOHYDRATES

oligosaccharidases.

Sucrase-a-Dextrinase D e f i c i e n c y . This h y b r i d enzyme has been found t o be markedly depressed o r absent i n about 100 docu mented cases (37-40). The malady appears t o be i n h e r i t e d as an autosomal r e c e s s i v e . A l l p a t i e n t s w i t h sucrase d e f i c i e n c y appear to have markedly depressed l e v e l s of α-dextrinase when isomaltose i s used as the s u b s t r a t e , but some d e x t r i n a s e a c t i v i t y u s u a l l y p e r s i s t s . This f i n d i n g , coupled w i t h t h e f a c t that sucrase and α-dextrinase a r e d i s t i n c t p r o t e i n s (12), suggests that the p r i mary genetic defect c o n s t i t u t e s the a l e r a t i o n o r absence o f the sucrase subunit w i t h secondary r e d u c t i o n o f i t s α-dextrinase partner. Symptoms produced upon i n g e s t i o n of sucrase a r e i d e n t i c a l w i t h those discussed above f o r l a c t a s e d e f i c i e n c y . Amylopectin i s u s u a l l y w e l l t o l e r a t e d even though a p p r e c i a b l e q u a n t i t i e s o f α-dextrins a r e released w i t h i n the i n t e s t i n e . This may r e f l e c t the r e l a t i v e l y l a r g e molecular weight o f the α-dextrins o r the f a c t that a p p r o p r i a t e b a c t e r i a i n colon may not be present i n s u f f i c i e n t numbers to metabolize them to small o s m o t i c a l l y a c t i v e fragments; a l s o , some h y d r o l y s i s of these α-dextrins may occur by a c t i o n of the r e s i d u a l α-dextrinase subunits o r perhaps other surface α-glucosidases such as glucoamylase. Treatment o f Disaccharidase

Deficiencies

Although i t appears t o be p o s s i b l e t o administer enzymes along w i t h a d i e t a r y o l i g o s a c c h a r i d e t o promote d i g e s t i o n , t h e expenseusually makes t h i s i m p r a c t i c a l . By f a r t h e simplest form of therapy i s the e l i m i n a t i o n o f the o f f e n d i n g carbohydrate s i n c e no s i n g l e carbohydrate c o n s t i t u t e s an o b l i g a t e source o f c a l o r i e s . References

1. Miller, D. and Crane, R.K. Biochim. Biophys. Acta (1961) 52:281-293. 2. Eichholz, A. and Crane, R.K. J. Cell Biol. (1965) 26:687-691. 3. Maestracci, D., Schmitz, J., Preiser, H. and Crane, R.K. Biochim. Biophys. Acta (1973) 323:113-124. 4. Hollingsworth, D.F. and Greaves, J.P. Am. J. Clin. Nutr. (1967)20:65-72. 5. Roberts, P.J.P. and Whelan, W.J. Biochem. J. (1960) 76: 246-253. 6. Bines, B.J. and Whelan, W.J. Biochem. J. (1960) 76:253-263. 7. Robyt, J.F. and French, D. J. Biol. Chem. (1970) 245:39173927. 8. Ugolev, A.M. Physiol. Rev. (1965) 45:555-595. 9. Fogel, M.R. and Gray, G.M. J. Appl. Physiol. (1973) 35: 263-267.



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Oligosaccharidases

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10. Gray, G.M. and Santiago, Ν.A. J. Clin. Invest. (1969) 48: 716-728. 11. Kelly, J.J. and Alpers, D.H. Biochim. Biophys. Acta (1973) 315:113-120. 12. Conklin, K.A., Yamashiro, K.M. and Gray, G.M. J. Biol. Chem. (1975) in press. 13. Lorenz-Meyer, Η., Blum, A.L., Haemmerli, H.P. and Semenza, G. Europ. J. clin. Invest. (1972) 2: 326-331. 14. Eggermont, E. and Hers, H.G. Eur. J. Biochem. (1969) 9:488496. 15. Dahlqvist, A. and Lindberg, T. Clin. Sci. (1966) 30:517. 16. Rosensweig, N.S. and Herman, R.H. J. Clin. Invest. (1968) 47:2253-2262. 17. Deren, J.J., Broitman, S.A., Zamcheck, N. J. Clin. Invest. (1967) 46:186-195. 18. Das, B.C. and Gray, G.M. Clin. Res. (1970) 18:378. 19. Das, B.C. and Gray, G.M. In preparation. 20. Alpers, D.H. and Cote, M.N. Amer. J. Phys. (1971) 221:865868. 21. Gray, G.M. and Ingelfinger, F.J. J. Clin. Invest. (1966) 45:388-398. 22. Gray, G.M. and Santiago, N.A. Gastro. (1966) 51-489-498. 23. Bayless, T.M. and Rosensweig, N.S. J. Am. Med. Assoc. (1966) 197:968. 24. Huang, S.-S. and Bayless, T.M. Science (1968) 160:83. 25. Newcomer, A.D. and McGill, D.B. Gastroenterology (1967) 53:881. 26. Littman, Α., Cady, A.B., Rhodes, J. Is. J. Med. Sci. (1968) 4:110. 27. Gudmand-Höyer, E., Dahlqvist, Α., Jarnum, S. Scand. J. Gastroenterol. (1969) 4:377. 28. Sheehy, T.W. and Anderson, P.R. Lancet (1965) 2:1. 29. Welsh, J.D., Rohrer, V., Knudsen, K.B., Paustian, F.F. Arch. Int. Med. (1967) 120:261. 30. Littman, Α., Cady, A.B., Rhodes, J. Is. J. Med. Sci.(1968) 4:110. 31. Cook, G.C., Kajubi, S.K. Lancet (1966) 1:725. 32. Chung, M.H. and McGill, D.B. Gastro. (1968) 54:225. 33. Elliott, R.B., Maxwell, G.M., Vawser, N. Med. J. Aust. (1967) 1:46. 34. Davis, A.E. and Bolin, T. Nature (1967) 216:1244. 35. Desai, H.G., Chitre, A.V., Parekh, D.V., Jeejeebhoy, K.N. Gastro. (1967) 53:375. 36. Gilat, T., Kuhn, R., Gelman, E. and Mizrahy, O. Dig. Dis. (1970) 15:895-904. 37. Burgess, E.A., Levin, B., Mahalanabis, D., Tonge, R.E. Arch. Dis. Childhood (1964) 39:431. 38. Prader, A. and Auricchio, S. Ann. Rev. Med. (1965) 16:345. 39. Sonntag, W.M. et al. Gastro. (1964) 47:18.


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40. Auricchio, S. et al. J. Pediat. (1965) 66:555. 41. Gray, G.M. Annual Review Medicine (1971) 22:391-404.


*Research supported by USPHS Grant AM 11270 and Research Career Development Award AM 47443 from the N a t i o n a l I n s t i t u t e s o f Health.


Oligosaccharidases of the Small Intestinal Brush Border - ACS

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