Lysosomal Enzyme Deficiency DiseasesGlycoprotein Catabolism in

not bind to Concanavalin A-. Sepharose (24). A six year-old boy was reported by Patel et al ..... Opheim, D. J.; and Touster, 0. J Biol Chem, 252:...
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7 Lysosomal Enzyme Deficiency Diseases—Glycoprotein

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Catabolism in Brain Tissue ERIC G. BRUNNGRABER Missouri Institute of Psychiatry and the Departments of Psychiatry and Biochemistry, University of Missouri—Columbia, St. Louis, MO 63139 The heteropolysaccharide chains of the glycoproteins in brain tissue provide the glycosidic linkages which serve as the substrates for a number of lysosomal glycosidases. The genetically-induced absence of a glycosidase, or a mutation which causes one or more of these glycosidases to become non-functional, produces a block in the catabolism of these protein­linked heteropolysaccharides. This pathological event, observed in the lysosomal enzyme deficiency diseases, causes an accumulation of undegraded glycopeptides or oligosaccharides, and an excessive urinary excretion of these substances. About 65 percent of the glycoprotein-carbohydrate of brain tissue is released in the form of sialoglycopeptides which contain NeuNAc, Fuc, GlcNAc, Gal, and Man (1) after treatment of the glycoproteins with proteolytic enzymes, usually papain or pronase. Approximately 20-40 percent of these sialoglycopeptides contain, in addition, sulfate ester groups. All of these sialoglycopeptides appear to contain 3 Man residues per glycopeptide molecule (Table I). Two, three, and possibly four chains consisting of -GlcNAc-Gal-NeuNAc (Fuc) are attached to this trimannoside core. The sialoglycopeptides were partially resolved by column electrophoresis. Fraction I contains the sialoglycopeptide with the highest molecular size and negative charge. The molecular size TABLE I SIALOGLYCOPEPTIDES DERIVED FROM BRAIN GLYCOPROTEINS Fraction: I II III IV V VI VII NeuNAc 4 4 3 2 1 1 3 Gal 4 4 4 3 3 2 2 Man 3 3 3 3 3 3 3 GlcNAc 6 6 6 6 5 4 4 Fuc 1 1 1 1 2 1-2 1-2 0-8412-0452-7/78/47-080-135$05.00/0 © 1978 American Chemical Society

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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

and charge decreased as the number and s i z e of the - G l c N A c - G a l NeuNAc (or Fuc) chains attached to the trimannoside core i s r e ­ duced. The composition of f r a c t i o n s VI and VII i s of p a r t i c u l a r interest. These s i a l o g l y c o p e p t i d e s account for about 15-20 p e r ­ cent of the glycopeptide-carbohydrate of whole r a t b r a i n . Two -GlcNAc-Gal-NeuNAc (or Fuc) chains are attached to the trimanno­ side core. The Man residues are s u f f i c i e n t l y exposed so that these glycopeptides show a greater a f f i n i t y to Con A-Sepharose columns (2) than the more a c i d i c p o l y s i a l o g l y c o p e p t i d e s of f r a c ­ t i o n s I to IV. These glycopeptides a l s o have a greater a b i l i t y to i n h i b i t the p r e c i p i t a t i o n of glycogen by Concanavalin A than the more n e g a t i v e l y charged p o l y s i a l o g l y c o p e p t i d e s (3). I t was a l s o found (4) that these glycopeptides y i e l d m e t h y l a t i o n p r o ­ ducts that resemble those obtained from a glycopeptide from transferrin:

( A)

Gal+GlcNAc (1+2 ) Man (1+6 )

^ Man+Gl cN Ac+Gl cN Ac-*As η Gal+GlcNAc (1+2 ) Man (1+3 ) '

S i a l o g l y c o p e p t i d e s of f r a c t i o n s I to V , c h a r a c t e r i z e d by higher values f o r the r a t i o s of NeuNAc/Man, Gal/Man, and GlcNAc/ Man may possess s t r u c t u r e s which resemble those of the g l y c o ­ peptides d e r i v e d from f e t u i n ( 4 ) , as suggested some years ago (5) and f o r which Krusius et a l C4,6) have obtained some e v i d e n c e . : Gal-GlcNAc^ Man Gal-GlcNAc (B)

\

x

Gal-GlcNAc^ Gal-GlcNAc'"

Man'

/

Man-GlcNAc- Gl cN Ac -Agn

The b r a i n s i a l o g l y c o p e p t i d e s contained r e l a t i v e l y high amounts of t e r m i n a l nonsubstituted G a l and GlcNAc residues which suggested that the p e r i p h e r a l chains are often incomplete (6). As we s h a l l see, an accumulation of glycopeptides with s t r u c t u r e s corresponding to (A) have been shown to accumulate i n f u c o s i d o s i s , I - c e l l d i s e a s e , and the GM1- and GM2-gangliosidoses. An accumulation of o l i g o s a c c h a r i d e s or glycopeptides with 3 or 4 t e r m i n a l -GlcNAc-Gal-NeuNAc (or fucose) branches has not yet been r e p o r t e d . Does t h i s suggest the presence i n t i s s u e s of a h i t h e r t o undescribed endo-g-glucosaminidase which s p l i t s the bond between the GlcNAc r e s i d u e of the -GlcNAc-Gal-NeuNAc(or Fuc) branch chain l i n k e d to the trimannoside core? The existance of such an enzyme might e x p l a i n the r e l a t i v e l y l a r g e accumulation of o l i g o s a c c h a r i d e s and glycopeptides with s t r u c t u r e (A) i n the

7.

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Enzyme

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Diseases

137

l i v e r , b r a i n and u r i n e i n s e v e r a l of the lysosomal enzyme d e f i ­ ciency d i s e a s e s , s i n c e t h i s s t r u c t u r e would be the accumulated end product of the catabolism of a wide v a r i e t y of h e t e r o p o l y saccharide c h a i n s . Finne et a l (7) demonstrated the presence of d i s i a l o s y l (a-N-acetylneuraminyl-(2+8)N-acetylneuraminyl) groups i n the s i a l o g l y c o p e p t i d e s from b r a i n . Thus, glycopeptides of f r a c t i o n s I and I I , which contain 4 NeuNAc r e s i d u e s , may c o n t a i n d i ­ s i a l o s y l groups, ensuring the a v a i l a b i l i t y of one or two of the 4 Gal residues as a t e r m i n a l sugar or as a sugar that i s p e n u l t i ­ mate to a t e r m i n a l fucose or s u l f a t e r e s i d u e . Approximately 20-25 percent of the g l y c o p r o t e i n - c a r b o h y d r a t e of r a t b r a i n c o n s i s t e d of mannoglycopeptides which c o n t a i n 6 Man and 2 GlcNAc residues per glycopeptide molecule (8). Manno­ glycopeptides , r e a d i l y i s o l a t e d since they bind s t r o n g l y to Con A-Sepharose, contained t e r m i n a l Man residues and would not be a f f e c t e d i n the G M l - g a n g l i o s i d o s i s and fucosidoses. They would make a major c o n t r i b u t i o n to the accumulated o l i g o s a c c h a ­ r i d e s i n mannosidosis. When p u r i f i e d l i v e r lysosomes were incubated i n the presence of f e t u i n or orosomucoid ( 9 ) , about one h a l f of the peptide bonds were cleaved and most of the t e r m i n a l NeuNAc residues were r e ­ leased. Release of Gal and GlcNAc was slower and d i d not exceed 30 percent of the t h e o r e t i c a l y i e l d . Release of Man was not d e t e c t e d , and branch p o i n t s i n which Man r e s i d u e s were i n v o l v e d appeared to r e s i s t a t t a c k . Many s t u d i e s have shown that p u r i f i e d 3-galactosidase and β - Ν - a c e t y l g l u c o s a m i n i d a s e can cleave t e r m i n a l l y exposed c a r b o ­ hydrate groups of i n t a c t g l y c o p r o t e i n s , and i t appears l i k e l y that t h i s process occurs i n the lysosomes of the i n t a c t organism. There may be an e x c e p t i o n , however. H i g h l y p u r i f i e d a-fucosidase from r a t l i v e r c a t a b o l i z e d the h y d r o l y s i s of 1-2, 1-3, and 1-4 f u c o s y l linkages i n g l y c o p e p t i d e s , but not i n i n t a c t g l y c o ­ p r o t e i n s (10). Since fucose i s i n a t e r m i n a l l y l i n k e d p o s i t i o n , f a i l u r e to cleave i t w i l l prevent the f u r t h e r degradation of any chains terminated by t h i s sugar. An endo-3-N-acetylglucosaminidase which hydrolyzes the d i - N , N - a c e t y l c h i t o b i o s y l bond i n the l i n k a g e r e g i o n i n g l y c o ­ peptides to produce o l i g o s a c c h a r i d e s w i t h GlcNAc i n a reducing p o s i t i o n and Asn-GlcNAc ( F i g . 2) has been demonstrated i n the hen oviduct (11) and methods f o r i t s assay have been described (12). Although most of the carbohydrate can be removed from n a t i v e g l y c o p r o t e i n s by the endo-glucosaminidase, denaturation of the g l y c o p r o t e i n s was necessary to e f f e c t complete renewal (13). The a c t i v i t y of t h i s enzyme accounts f o r the accumulation of o l i g o s a c c h a r i d e s i n some of the lysosomal enzyme d e f i c i e n c y diseases. O l i g o s a c c h a r i d e s may a l s o be produced by the a c t i o n of 4 - L - a s p a r t y l g l y c o s y l a m i n e amidohydrolase, which cleaves the bond between GlcNAc and asparagine (14). The s i z e and s t r u c t u r e of

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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

the carbohydrate group d i d not appear to be an important f a c t o r i n c o n t r o l l i n g a c t i v i t y , but the presence of amino a c i d s i n a d d i t i o n to the asparagine i n the peptide p o r t i o n of the g l y c o peptide molecule reduced the r a t e of t h i s r e a c t i o n . T h i s enzyme i s not a c t i v e on i n t a c t g l y c o p r o t e i n s (15). Treatment of the d e l i p i d a t e d b r a i n t i s s u e w i t h p r o t e o l y t i c enzymes r e l e a s e d a l l of the heteropolysaccharide chains of the g l y c o p r o t e i n s , along with glycosaminoglycans and n u c l e i c a c i d s . The l a t t e r substances are removed by p r e c i p i t a t i o n with c e t y l p y r i d i n i u m c h l o r i d e . D i a l y s i s of the glycopeptide p r e p a r a t i o n separates the s i a l o g l y c o p e p t i d e s , which are l a r g e l y n o n - d i a l y z a b l e , from the d i a l y z a b l e , Con-A-binding, mannoglycopeptides. In the storage d i s e a s e s , accumulated degradation products d e r i v e d from the s i a l o g l y c o p e p t i d e s w i l l appear i n the d i a l y z a b l e glycopeptide f r a c t i o n . Thus, an i n c r e a s e i n the carbohydrate content of t h i s f r a c t i o n does not n e c e s s a r i l y i n d i c a t e an ac­ cumulation of the normal c o n s t i t u e n t s of t h i s f r a c t i o n , but does i n d i c a t e the appearance of abnormal degradation products ( u s u a l l y present only i n t r a c e s ) d e r i v e d from the n o n d i a l y z a b l e sialoglycopeptides. Treatment of the d e l i p i d a t e d t i s s u e with p r o t e o l y t i c enzymes has the disadvantage of obscuring the evidence f o r the p o s s i b l e accumulation of glycopeptides or o l i g o s a c c h a r i d e s i n the t i s s u e . These substances can be recovered q u i t e simply by e x t r a c t i n g the t i s s u e with water or aqueous s o l v e n t s . As we s h a l l see, the l a t t e r procedure has the advantage of p r o v i d i n g the accumulated o l i g o s a c c h a r i d e s which have proved to be amenable f o r s e p a r a t i o n and s t r u c t u r a l a n a l y s i s . A thorough study would allow f o r the i s o l a t i o n of these substances p r i o r to s u b j e c t i n g the i n s o l u b l e t i s s u e r e s i d u e to p r o t e o l y s i s . GM2-gangliosidoses ( d e f i c i e n c y of β - h e x o s a m i n i d a s e ) . Tingey (16) reported that the hexosamine l e v e l i n the de­ l i p i d a t e d b r a i n t i s s u e from Tay-Sachs diseased p a t i e n t s was elevated. Hexosamine-containing substances c o u l d a l s o be ex­ t r a c t e d from the residue w i t h b o i l i n g water. While only 3-10 percent of the t o t a l b r a i n hexosamine could be e x t r a c t e d from normal b r a i n i n t h i s manner, the amount of the m a t e r i a l extracted from Tay-Sachs b r a i n was s u b s t a n t i a l l y e l e v a t e d . An e l e v a t i o n of protein-bound hexosamine i n Tay-Sachs disease was confirmed by others (17-21). Suzuki (20) found that the aqueous e x t r a c t prepared from the b r a i n of p a t i e n t s with Sandhoff's disease contained elevated l e v e l s of hexosamine while the i n c r e a s e i n hexosamine c o n c e n t r a t i o n of defatted Tay-Sachs b r a i n t i s s u e and i n aqueous e x t r a c t s prepared from the residue was moderate. Glycopeptides were recovered from the d e l i p i d a t e d c e r e b r a l c o r t i c a l t i s s u e by treatment of the m a t e r i a l w i t h papain (22-24). The NeuNAc content of the d i a l y z a b l e and n o n - d i a l y z a b l e g l y c o ­ peptides obtained from Tay-Sachs b r a i n s was normal. Thus, catabolism of these heteropolysaccharide chains d i f f e r e d from that of the g a n g l i o s i d e s ( F i g . 1 ) : cleavage of the NeuNAc residues

7.

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Enzyme

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Diseases

139

was not dependent on the p r i o r removal of hexosamine. The conc e n t r a t i o n and carbohydrate composition of the n o n - d i a l y z a b l e s i a l o g l y c o p e p t i d e s d i d not d i f f e r from normal v a l u e s . On the other hand, a f o u r - f o l d e l e v a t i o n of hexosamine and mannose content i n the d i a l y z a b l e glycopeptides f r a c t i o n was observed i n both Tay-Sachs and Sandhoff's d i s e a s e s . The accumulated d i a l y z a b l e glycopeptides d i d not bind s t r o n g l y to Con A-Sepharose and presumably d i d not contain the t e r m i n a l Man linkages which i n t e r a c t with the l e c t i n . Thus, the accumulated m a t e r i a l d i d not correspond to the mannoglycopept i d e s , also present i n the d i a l y z a b l e glycopeptide p r e p a r a t i o n . Probably breakdown products of the s i a l o g l y c o p e p t i d e s , these accumulated glycopeptides presumably contained t e r m i n a l GlcNAc residues. Oligosaccharides which contained a trimannosyl core and GlcNAc at non-reducing t e r m i n i were i s o l a t e d from the l i v e r of a case with Sandhoff's disease (25). Accumulation of t h i s GlcNAc-containing o l i g o s a c c h a r i d e , or the excessive e x c r e t i o n i n the u r i n e , was not observed i n Tay-Sachs disease (26). The o l i g o s a c c h a r i d e s were i s o l a t e d by homogenizing the l i v e r i n 10 volumes of chloroform-uiethanol-water ( 1 : 2 : 0 . 3 , v / v / v ) . The m a t e r i a l was then p a r t i t i o n e d i n t o an upper aqueous phase which was evaporated to dryness. Gangliosides were extracted from the d r i e d residue by repeated e x t r a c t i o n with dry chloroformmethanol. Glycopeptides were a l s o i s o l a t e d from d e l i p i d a t e d t i s s u e by means of p r o t e o l y s i s with papain. Tsay and Dawson (27) minced b r a i n t i s s u e i n water. The mixture was sonicated and c e n t r i f u g e d , and the water s o l u b l e m a t e r i a l was f r a c t i o n a t e d by g e l f i l t r a t i o n . The s t r u c t u r e of an i s o l a t e d o l i g o s a c c h a r i d e was determined by treatment with the appropriate g l y c o s i d a s e s , periodate o x i d a t i o n , and GLC analysis. Its s t r u c t u r e corresponded to that of the o l i g o saccharide p r e v i o u s l y i s o l a t e d from the l i v e r of Sandhoff p a t i e n t s (25). The amount i s o l a t e d from b r a i n was extremely small compared to the l e v e l s accumulated i n the l i v e r of these patients. GlcNAc (3,1+2) Man (a, 1+3). GlcNAc (3,1+2)Man (a, 1+6)

Man (3,1-^4) GlcNAc

Whether the o l i g o s a c c h a r i d e s i n b r a i n are a product of b r a i n metabolism i s not e n t i r e l y c l e a r . It i s p o s s i b l e that incompletely degraded m a t e r i a l s produced i n the l i v e r and organs can enter the c i r c u l a t i o n from which they may be taken up by brain c e l l s . On the other hand, b r a i n t i s s u e does appear to contain heteropolysaccharide chains (Table I) with a carbohydrate composition which corresponds to that of the accumulated product.

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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

The d e f i c i e n c y of hexosaminidase A i n Tay-Sachs d i s e a s e , and hexosaminidases A and Β i n Sandhoff f s disease r e s u l t s i n the accumulation of g a n g l i o s i d e GM2 ( F i g . 1) and o l i g o s a c c h a r i d e s ( F i g . 2, r e a c t i o n D) both of which c o n t a i n t e r m i n a l l y l i n k e d β-hexosamine r e s i d u e s . Both hexosaminidases A and Β have been shown to act on t e r m i n a l N - a c e t y l glucosamines of o l i g o s a c c h a ­ r i d e s derived from g l y c o p r o t e i n s (28). G M l - g a n g l i o s i d o s i s , d e f i c i e n c y of F - g a l a c t o s i d a s e . An o l i g o s a c c h a r i d e c o n s i s t i n g of a degradation product d e r i v e d from g l y c o p r o t e i n s was i s o l a t e d from the l i v e r of p a ­ t i e n t s with G M l - g a n g l i o s i d o s i s (29): Gal(β,1+4)GlcNAc(β, 1+2)Man(α,1+3)^ Gal (β, 1+4) GlcNAc (β, 1+2 )Man (α, 1+6 )

'

Man(β,1+4)GlcNAc

The n o n d i a l y z a b l e and d i a l y z a b l e glycopeptides recovered a f t e r d i g e s t i o n of the d e l i p i d a t e d residue from the c e r e b r a l gray matter showed an over two-fold e l e v a t i o n i n Gal content (31,32). Man and GlcNAc were a l s o e l e v a t e d . These r e s u l t s suggested the accumulation of glycopeptides with exposed Gal residues. Degradation can proceed no f u r t h e r a f t e r the removal of the t e r m i n a l NeuNAc and fucose r e s i d u e s . There was an es­ p e c i a l l y marked accumulation of glycopeptides which were d e r i v e d from the NeuNAc-rich, h i g h l y n e g a t i v e l y charged, glycopeptides the sugar content of which was c h a r a c t e r i z e d by h i g h values f o r the Gal/Man and GlcNAc/Man r a t i o s ( f r a c t i o n s I and I I , Table I ) . In a d d i t i o n to these changes, e l e c t r o p h o r e t i c a n a l y s i s of the n o n - d i a l y z a b l e glycopeptide p r e p a r a t i o n suggested that a l a r g e p r o p o r t i o n of the s i a l o g l y c o p e p t i d e s were d e f i c i e n t i n -GlcNAcGal-NeuNAc branches which are attached to the trimannoside c o r e . This change cannot be a t t r i b u t e d to the absence of β - g a l a c t o s i dase, and may represent secondary changes due to the e f f e c t s of the d i s e a s e . In a second case, the c o n c e n t r a t i o n of d i a l y z a b l e glycopeptides c o n t a i n i n g hexosamine, Gal and Man showed a 3-7 f o l d e l e v a t i o n which was dependent on the b r a i n area s t u d i e d . The accumulated glycopeptides d i d not bind to Concanavalin A Sepharose (24). A s i x y e a r - o l d boy was reported by P a t e l et a l (33) to have a 2.5 f o l d e l e v a t i o n i n g l y c o p r o t e i n - g a l a c t o s e i n brain. Tsay and Dawson (27) recovered an o l i g o s a c c h a r i d e from the b r a i n of G M l - g a n g l i o s i d o s i s p a t i e n t s the s t r u c t u r e of which was i d e n t i c a l to that recovered from l i v e r and u r i n e by Wolfe et a l (29,30,31). I t s s t r u c t u r e resembled that of the o l i g o s a c c h a r i d e s recovered from the b r a i n of Sandhoff p a t i e n t s , except for the two t e r m i n a l l y - l i n k e d Gal residues ( F i g . 2 ) . Only a r e l a t i v e l y small amount of t h i s m a t e r i a l was recovered from b r a i n ; the accumulation of g a n g l i o s i d e GM1 and glycopeptides released by p r o t e o l y s i s was c o n s i d e r a b l y g r e a t e r .

Figure

G M 3

GM2

GM1

^-glucosidase

galactosidase

1. Block in ganglioside

$

neuraminidase

^hexosaminidase

/ί-galactosidase

BLOCKED

+ Glc

GM2

Gal

GM1

BLOCKED

IN

DISEASE

GM2-gangliosidosis, and

GAUCHERS

>-Gal

+ Neu Ν A c

GANGLIOSIDOSIS

GANGLIOSIDOSIS

Gal

+ Gal Ν Ac

IN

+

IN

catabolism in GM-l-gangliosidosis, Gauchers Disease

Cer

Cer-GIc

Cer-Glc-Gal

NeuNAc

J 1 i— I

Cer-Glc-Gal

-BLOCKED

— I

-NeuNAc

I

Cer-Glc-Gal-GalNAc

•

2)NeuNAc

1

C e r ( 1 « — 1 . β ) G l c ( 4 « - 1 . β) G a l ( 4 « - 1 , ^ g ) G a l Ν A c ( 3 * - 1 . β)

S «s:

Ci

Ο > w Η

142

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

Fuc F u c - G a l - GlcNAc - M a n

1

M a n - G l c N A c —GlcNAc - A s n Fuc-Gal - GlcNAc-Man A

^ (Endo-0-N-glucosaminidase)

^

Fuc-Gal-GlcNAc-Man

Fuc \ Man —GlcNAc

I GlcNAc-Asn

+

Fuc- Gal-GlcN Ac-Man Β

^

Gal — G l c N A c —Man ΓM a n — G l c N A c

+

Fuc

Gal — G l c N A c — M a n ' C

\

(Qt-Fucosidase)

(Ot-Fucosida idase)

G l c N A c - A s n + Fuc N-aspartyl-

^

glucosaminidase

( β-Galactosidase)

GlcNAc

+ Asn

GlcNAc — Man

\

G l c N A c —Man

/

Man —GlcNAc

+ Gal

( β-hexosaminidase)

i

Man, Man

>

Man

(Q£

1

-GlcNAc

Man

GlcNAc

mannosidase)

I an —GlcNAc M

(β

+

+ Man

mannosidase) +

GlcNAc

Figure 2. Probable catabolic pathway for the degradation of oligosaccharide chains derived from brain glycoproteins. Reactions B, C, D, and Ε are blocked in fucosidosis, GMl-gangliosidosis, GM2-gangliosidosis, and mannosidosis, respec­ tively.

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Gaucher*s Disease ( d e f i c i e n c y of glucocerebrosidase) Whether p a r t i a l degradation products derived from g l y c o ­ p r o t e i n s accumulate i n the b r a i n of the neuropathic forms of Gaucher s disease i s not known. Kanfer et a l (34) t r e a t e d de­ f a t t e d l i v e r t i s s u e with p r o t e o l y t i c enzymes. Glycopeptides d e r i v e d from g l y c o p r o t e i n s were markedly elevated i n the Gaucher l i v e r ; a 20 f o l d e l e v a t i o n i n hexose, hexosamine, fucose and NeuNAc was recorded. The accumulated m a t e r i a l contained Gal and GlcNAc as the major sugars; Glc and Man were present i n t r a c e s . The disease i s caused by d e f i c i t i n glucocerebrosidase, a 3-glucosidase. Although glucose-containing g l y c o p r o t e i n s un­ doubtedly e x i s t (1), there i s no evidence f o r the accumulation of G l c - c o n t a i n i n g glycopeptides or o l i g o s a c c h a r i d e s . Gaucher spleen appears to produce an excess of a g l y c o p r o t e i n a c t i v a t i n g f a c t o r , capable of a c t i v a t i n g the glucocerebrosidase from normal spieen (35-37)· While the a c t i v a t o r from Gaucher spleen was a g l y c o p r o t e i n , that from c o n t r o l spleens was not. The amino a c i d composition of the two a c t i v a t o r s a l s o d i f f e r e d (38). The r o l e of the g l y c o p r o t e i n a c t i v a t o r remains obscure. Metachromatic Leukodystrophy (MLD) and M u l t i p l e S u l f a t a s e D e f i c i e n c y (MSP). 1

The s i a l o g l y c o p e p t i d e p r e p a r a t i o n from b r a i n contains mole­ cules which c a r r y s u l f a t e - e s t e r groups. Consequently, i t ap­ peared l i k e l y that p a r t i a l degradation products derived from g l y c o p r o t e i n s which bear s u l f a t e d o l i g o s a c c h a r i d e chains might accumulate i n MLD, a disease caused by the absence of a f u n c t i o n ­ ing s u l f a t a s e A. However, s u l f a t i d e s are the n a t u r a l substrate f o r t h i s enzyme. The hexosamine l e v e l s i n the d e l i p i d a t e d t i s s u e residue from cases of MLD, i n which i t was e s t a b l i s h e d that s u l f a t a s e A i s d e f i c i e n t , f e l l w i t h i n the normal range (39,40). The s i t u a t i o n d i f f e r s i n MSD, a v a r i a n t of MLD i n which the t i s s u e s of the p a t i e n t s l a c k s u l f a t a s e s A, B, and C. The non­ l i p i d hexosamine i n t i s s u e s from such p a t i e n t s show an e l e v a t i o n of protein-bound hexosamine (41), and i t has been shown that t h i s i s due, at l e a s t i n p a r t , by the e l e v a t i o n of glycosaminoglycans· I t i s not known whether glycopeptides or o l i g o s a c c h a r i d e s de­ r i v e d from g l y c o p r o t e i n s accumulate i n t h i s d i s e a s e . I t has been suggested (42) that O-sulfated glycosaminoglycans are the n a t u r a l substrate f o r s u l f a t a s e B, s i n c e t h i s enzyme i s d e f i c i e n t i n two diseases (MSD and Maroteaux-Lamy disease) i n which O-sulfated glycosaminoglycans accumulate (mainly dermatan s u l f a t e ) . However, c o - c u l t i v a t i o n of MSD f i b r o ­ b l a s t s with S a n f i l i p p o A or Hunter c e l l s d i d not c o r r e c t f o r the degradation defect i n glycosaminoglycan-SO/j-S metabolism. S a n f i l i p p o and Hunter f i b r o b l a s t s l a c k heparan s u l f a t a s e and dermatan s u l f a t a s e , but a r y l s u l f a t a s e Β i n these c e l l s i s normal or elevated (42). These f i n d i n g s l e d to the suggestion that s u l f a t a s e Β may be i n v o l v e d i n degrading s u l f a t e d glycopeptides (43). 35

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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

S u l f a t a s e Β appears to correspond to N-acetylgalactosamine 4 - s u l f a t a s e (44). The s u l f a t a s e r e s p o n s i b l e f o r the accumula­ t i o n of protein-bound hexosamine s t i l l remains to be i d e n t i f i e d . Nor i s i t yet e s t a b l i s h e d whether s u l f a t e d glycopeptides d e r i v e d from g l y c o p r o t e i n s accumulate i n MSD. The unknown s u l f a t a s e may be a component of s u l f a t a s e A or B, both of which e x i s t i n m u l t i p l e forms, or a s u l f a t a s e which does not respond to the s y n t h e t i c substrates commonly i n use to detect s u l f a t a s e a c t i " vity. The mucopolysaccharidoses. The defects i n H u r l e r , Hunter, and S a n f i l i p p o A and Β syndromes, those forms of the mucopolysaccharidoses i n v o l v i n g severe n e u r o l o g i c a l d e f i c i t s , are caused by nonfunctioning α - i d u r o n i d a s e , s u l f o i d u r o n a t e s u l f a t a s e , heparan-N-S-sulfatase, and Ofr-N-acetylglucosaminidase, r e s p e c t i v e l y . These linkages a r e , as f a r as i s known, absent i n g l y c o p r o t e i n s . Consequently, no a b e r r a t i o n i n g l y c o p r o t e i n catabolisrc i s expected, nor has such a defect been r e p o r t e d . Glycopeptides i s o l a t e d from the b r a i n of a Hunter p a t i e n t showed no remarkable changes ( 4 5 ) · Diseases with a primary l e s i o n i n g l y c o p r o t e i n c a t a b o l i s m . Mannose-rich o l i g o s a c c h a r i d e s accumulate i n the b r a i n of mannosidosis p a t i e n t s (46-48): Man-Man-Gl cNAc? Man-Man-Man-Gl cNAc Man-Man

^Man-GlcNAc

Man' The disease occurs a l s o i n Angus c a l v e s , and the defatted b r a i n t i s s u e residue of the a f f l i c t e d animals showed a 4-6 f o l d e l e v a ­ t i o n i n Man content (49). D e f i c i e n c y of α - m a n n o s i d a s e causes a b l o c k i n the catabolism of the h e t e r o p o l y s a c c h a r i d e c h a i n s : mannose-containing glycopeptides or o l i g o s a c c h a r i d e s are d e r i v e d from the s i a l o g l y c o p e p t i d e trimannoside c o r e , as w e l l as from the mannoglycopeptides· In p a t i e n t s with a d e f i c i e n c y of ot-fucosidase (fucosidosis), f u c o s e - c o n t a i n i n g substances were shown to accumulate i n the d e l i p i d a t e d t i s s u e , i n c l u d i n g that of the b r a i n (50). Aqueous e x t r a c t s of sonicated b r a i n t i s s u e y i e l d e d an o l i g o s a c c h a r i d e (27) : Fuc-Gal-GlcNAc-Man V

Man-GlcNAc Fuc-Gal-GfccNAc-Man^

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A disaccharide, fucosylated N-acetylglucosamine, was also de­ tected i n the brains of fucosidosis patients (Fig. 2). Aspartylglucosaminuria i s caused by a d e f i c i t i n the enzyme l-aspartamido-3-N-acetylglucosamine amidohydrolase. Aspartylglucosaminylamine and aspartyloligosaccharides accumulate i n organs, including the brain (51,52). The mucolipidoses are caused by a deficiency of α-neuramini­ dase (53) resulting i n the excessive excretion of s i a l y l o l i g o saccharides. Most of the oligosaccharides isolated had a core consisting of three mannose residues to which two GlcNAc-GalNeuNAc chains, or incomplete portions of these chains, were attached (54). Although not a l l of the linkages i n the oligo­ saccharides of brain tissue have been definitively established, i t appears l i k e l y that their structure w i l l prove to be identi­ cal to that of one of the sialoglycopeptides isolated from urine (55): NeuNAc ( α , 2+6) Gal ( β , 1+4)GlcNAc ( β , 1+2)Man (α, 1+3\

/

Man(β,1+4)GlcNAc

NeuNAc ( α , 2+6 ) Gal ( 3,1+4 ) GlcNAc ( β , 1+2 )Man (α, 1+6 )' A sequence of the steps involved i n the catabolism of the heteropolysaccharides of brain glycoproteins has been postulated (Fig. 2) based on the structures of the oligosaccharides which accumulate i n the various lysosomal enzyme diseases discussed above. Niemann-Pick's Disease The primary lesion i n the i n f a n t i l e , type A, form of Nie­ mann Pick's disease i s a deficiency of the enzyme, sphingomye­ linase. While sphingomyelin i s the primary storage product, many cases which have been examined have demonstrated an increase i n gangliosides and cholesterol i n brain tissue. Tingey (16) and Norman et a l (56) have reported an elevation of proteinbound hexosamine as well. Brunngraber et a l (57) noted a two fold elevation of glycoproteins-carbohydrate which was recovered as sialoglycopeptides upon proteolytic digestion of the d e l i p i dated tissue residue from gray matter. Mannoglycopeptide levels remained within the normal range. The composition of the s i a l o ­ glycopeptides was altered, since the number of -GlcNAc-GalNeuNAc chains attached to the internal trimannoside core was reduced. I t i s possible, but not demonstrated, that the accumu­ lated glycopeptides i n Niemann-Pick disease may correspond to the sialylated forms of the oligosaccharides which accumulate i n the GMl-gangliosidoses. I t was suggested that sphingomyelin catabolism may be an early step i n the degradation of plasma mem­ branes, and a lesion i n the catabolism of this phospholipid may have the secondary effect of impeding the degradation of other plasma membrane components.

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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

Neuronal-Ceroid L i p o f u s c i n o s e s (NCF). A form of NCF may i n v o l v e g l y c o p r o t e i n storage. Adelman et a l (58) found a n e a r l y two f o l d i n c r e a s e i n the NeuNAc con­ tent of s i a l o g l y c o p e p t i d e p r e p a r a t i o n s obtained from t i s s u e sec­ t i o n s of the c e r e b r a l c o r t e x of a case with c l i n i c a l f e a t u r e s resembling those of Batten's d i s e a s e . C e l l r e c o g n i t i o n of lysosomal enzymes. Studies u t i l i z i n g c u l t u r e d f i b r o b l a s t s d e r i v e d from p a t i e n t s w i t h lysosomal enzyme d e f i c i e n c y diseases have shown that these c e l l s s e c r e t e lysosomal g l y c o s i d a s e s i n t o the medium, and are capable of r e - a s s i m i l a t i n g these enzymes. Hickman and Neuman (59,60) have proposed t h a t lysosomal enzymes are t r a n s p o r t e d from t h e i r s i t e of s y n t h e s i s and g l y c o s y l a t i o n i n the G o l g i apparatus to the lysosome by t h i s process of e x o c y t o s i s and subsequent p i n o c y t o t i c r e - i n c o r p o r a t i o n . Such a mechanism may be a p a r t of a membrane r e c y c l i n g mechanism, s i n c e i n t e r n a l i z a ­ t i o n of the enzyme i s accomplished by an i n t e r n a l i z a t i o n of a patch of plasma membrane, forming p i n o c y t o t i c v e s i c l e s d e s t i n e d to fuse with the lysosome (61). F i b r o b l a s t s from p a t i e n t s a f f l i c t e d with one of the l y s o s o ­ mal d e f i c i e n c y diseases are capable of a s s i m i l a t i n g the d e f i ­ c i e n t enzyme which had been added to the c u l t u r e medium, thereby c o r r e c t i n g the metabolic d e f e c t . Uptake of lysosomal g l y c o s i ­ dases i s dependent on the r e c o g n i t i o n of the h e t e r o p o l y s a c c h a r i d e c h a i n of the g l y c o p r o t e i n enzyme by a r e c e p t o r on the s u r f a c e of the f i b r o b l a s t . Thus, the t e r m i n a l Gal r e s i d u e appeared to be important f o r the r e c o g n i t i o n and uptake o f a-N-acetylglucosaminidase (62) by f i b r o b l a s t s of S a n f i l i p p o Β p a t i e n t s . Mannose residues appeared to be important f o r uptake of 3-galactosidase by f i b r o b l a s t s from p a t i e n t s with G M l - g a n g l i o s i d o s i s (63). Cultured f i b r o b l a s t s appear to recognize phosphohexosyl groups i n 3-glucuronidase, β-hexosaminidase, and 3-galactosidase (64,65) and a-L-iduronidase (66). The r e c o g n i t i o n of phospho­ hexosyl groups may be a general c h a r a c t e r i s t i c of p i n o c y t o s i s of lyosomal g l y c o s i d a s e s . These f i n d i n g s may be r e l a t e d to the e a r l i e r r e p o r t s on the i s o l a t i o n of a phosphoglycoprotein from b r a i n (67-69). Phosphorylated mannoglycopeptides were i s o l a t e d from the phosphoglycoprotein, and the phosphate r e s i d u e which i s attached to one of the h y d r o x y l groups of mannose, was removed by the a c t i o n of a l k a l i n e phosphatase (69). The r e l a t i o n of t h i s phosphoglycoprotein to the lysosomal g l y c o s i d a s e s remains to be e s t a b l i s h e d . I f the c a p a c i t y of the f i b r o b l a s t to s e c r e t e and r e - a s s i m i l a t e lysosomal enzymes i s a s p e c i f i c example of a more general phenomena, phosphoglycoproteins and/or g l y c o s i d a s e s may p l a y an important r o l e i n the turnover of the plasma membrane.

7.

BRUNNGRABER

1. 2. 3. 4. 5. 6. 7· 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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54. Michalski, J. C.; Strecker, G.; and Fournet, B. FEBS Lett, 79: 101, 1977. 55. Strecker, G.; Peers, M. C.; Michalski, J. C.; Hondi-Assah, T.; Fournet, B.; Spik, G.; Montreuil, J.; Farriaux, J. P.; Maroteaux, P.; and Durand, P. Eur J Biochem, 75: 391, 1977. 56. Norman, R. M.; Tingey, A. H.; and Fowler, M. C. Proc of the Fifth Internatl Congr Neuropathol, Zurich, 1965, Amster­ dam, Excerpta Med Found, 1966, p. 143. 57. Brunngraber, E. G.; Berra, B.; and Zambotti, V. Clin Chim Acta, 48: 173, 1973. 58. Adelman, L. S.; Young, E.; and Bass, Ν. H. Neurology, 24: 1045, 1974. 59. Hickman, S.; and Neufeld, E. F. Biochem Biophys Res Communs, 49: 992, 1972. 60. Neufeld, E. F.; Sando, G. N.; Sarvin, A. J.; and Rome, L. H. J Supramol Struct, 6: 95, 1977. 61. Brunngraber, E. G. Neurοchemistry of the Aminosugars, Springfield, Ill. C. C. Thomas, Pub. 1978. 62. Von Figura, Κ.; and Kresse, H. Prot Biol Fluids, 22: 275, 1975. 63. Hieber, V.; Distler, J.; Myerowitz, R.; Schmickel, R. D.; and Jourdian, G. W. Biochem Biophys Res Communs, 73: 710, 1966. 64. Kaplan, Α.; Fischer, D.; Achord, D.; and Sly, W. J Clin Invest, 60: 1088, 1977. 65. Kaplan, Α.; Achord, D. T.; and Sly, W. S. Proc Natl Acad Sci US, 74: 2026, 1977. 66. Sando, G. N.; and Neufeld, E. F. Cell, 12: 619, 1977. 67. Davis, L. G.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 30, 1976. 68. Davis, L. G.; Costello, A. J. R.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 35, 1976. 69. Davis, L. G.; Brettschneider, I.; and Brunngraber, E. G. Fed Proc, 36: 750, 1977. RECEIVED

March 15, 1978.