11 Glycoprotein Inhibitor of Lipoprotein Lipase from Aortic
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Intima—Its Possible Role in Atherosclerosis PREMANAND V. WAGH Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, AR 72206
Early observations on the presence of glycoproteins in the mammalian arterial wall have been reported (Buddecke, 1960; Muller -Spreer et a l , 1960; Berenson and Fishkin, 1962). Although considerable research interest is centered upon the relationship of glycoproteins in the aortic wall to atherosclerosis and ageing, little is known in regard to the structure and function of these macromolecules. Several soluble glycoproteins have been purified from bovine aorta after extraction of the tissue with neutral buffers (Radhakrishnamurthy et a l , 1964, Maier and Buddecke, 1971). The concentration of arterial glycoproteins in atherosclerosis and in ageing is a subject of question and is being actively investigated. The wet weight of the aorta has been found to be increased in atherosclerosis and among the aged (Manley and Mullinger, 1967). Our data (Wagh et a l , 1973), shown in Table I, provide evidence that the glycoprotein concentration increased significantly in atherosclerotic tissue as expressed by an increase in total neutral sugars, hexosamine and sialic acid. These results suggest that the increased glycoprotein concentration may have a functional role in the formation of atherosclerotic plaque. Several functions have been ascribed to arterial glycoproteins such as transplantation rejection and maintenance of structural integrity (Anderson, 1976). Furthermore, Ishii (1971) demonstrated that preparations of crude glycoprotein obtained from various organs including the aorta of dog inhibited dextran sulfate released lipoprotein lipase (LPL) activity of human plasma. LPL catalyzes the hydrolysis of the triglyceride moiety of chylomicrons and very low density lipoproteins (Eisenberg and Levi, 1975), and is normally located on the surface of endothelial cells where it is physiologically active in the normal clearance of lipoprotein-bound triglycerides (Robinson, 1970). Since the surface of the intimal layer of aortic wall is susceptive to continuous hemodynamic insult and thus may be i n volved in the genesis of atherosclerosis and other physiological events, we focussed our attention here for the purpose of isolation of glycoprotein. One of the shortcomings in the isolation 201 This chapter not subject to U . S . copyright Published 1978 A m e r i c a n C h e m i c a l Society
Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
of p u r i f i e d g l y c o p r o t e i n from the i n t i m a l region has been to separate i t from the r e s t of the a o r t i c w a l l . A new procedure (dermatome procedure) was developed i n our l a b o r a t o r y (Roberts et a l , 1974) that r a p i d l y and r e l i a b l y separates the intima i n s u f f i c i e n t q u a n t i t i e s from both porcine and human a o r t a . Studies on Porcine I n t i m a l G l y c o p r o t e i n ( L i p o l i p i n ) . We r e ported on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a novel g l y c o p r o t e i n from the i n t i m a l r e g i o n of porcine aorta (Wagh and Roberts 1972). Swine a o r t a was chosen because i n t h i s s p e c i e s , the aorta i s r e l a t i v e l y f r e e from a t h e r o s c l e r o t i c l e s i o n s and the m a t e r i a l i s r e a d i l y a v a i l a b l e . The p u r i f i c a t i o n procedure involved e x t r a c t i o n of i n t i m a l t i s s u e with n e u t r a l b u f f e r , (Nlfy^SO^ p r e c i p i t a t i o n between 40-90% s a t u r a t i o n and two DEAE-cellulose chromatographic steps. The p u r i f i e d g l y c o p r o t e i n was homogeneous upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n (4.86 S) and by polyacrylamide d i s c - g e l e l e c t r o p h o r e s i s . Generally, 40 mg of p u r i f i e d g l y c o p r o t e i n was obtained from 2 Kg of wet weight of t h o r a c i c a o r t a . Due to the presence of equimolar glucose and galactose and the absence of hydroxylysine i n the molecule, i t was suggested that t h i s g l y c o p r o t e i n was unique i n i t s c h a r a c t e r i s t i c s and t h e r e f o r e of a new type. The i s o e l e c t r i c p o i n t of the p u r i f i e d glycoprot e i n was 4.3 as observed by a n a l y t i c a l i s o e l e c t r i c focusing (Baig and Ayoub, 1976). Subsequent s t u d i e s (Roberts and Wagh, 1976) revealed that sodium dodecyl s u l f a t e - polyacrylamide g e l e l e c t r o p h o r e s i s of the n a t i v e and i t s S-carboxyamidomethyl d e r i v a t i v e a t d i f f e r e n t polyacrylamide concentrations d i d not a f f e c t the molecular weight (72,000 daltons) i n d i c a t i n g the absence of subunits. The carboxyt e r m i n a l amino a c i d was found to be s e r i n e . Attempts to determine the i d e n t i t y of the amino a c i d i n d i c a t e d that the amino group was not f r e e . The g l y c o p r o t e i n d i d not contain an a l k a l i - l a b i l e (0g l y c o s i d i c ) carbohydrate-peptide linkage as tested by $-eliminat i o n r e a c t i o n . The r e l e a s e of monosaccharides from the i n t a c t g l y c o p r o t e i n as a f u n c t i o n of time was studied employing m i l d a c i d h y d r o l y s i s (0.5 M HC1, 80°C) and a l s o by the use of neuraminidase, a-D- and $-D-glucosidases and $-D-N-acetylglucosaminidase. From the observations on the r e l e a s e of monosaccharides and analogy with standard features determined by other i n v e s t i g a t o r s on s o l u b l e a o r t i c g l y c o p r o t e i n s (Radhakrishnamurthy e t a l , 1964; Radhakushnamurthy and Berenson, 1966; Klemer and Nager, 1967; Maier and Buddecke, 1971), a p r e d i c t i o n was made as to the general features of the carbohydrate moiety of the g l y c o p r o t e i n ( F i g . 1 ) . This p o s t u l a t e d s t r u c t u r e must s t i l l withstand r e s u l t s of f u t u r e i n v e s t i g a t i o n s i n c l u d i n g methylation s t u d i e s , o x i d a t i o n with p e r i o date and the use of other s p e c i f i c g l y c o s i d a s e s . The p u r i f i e d porcine i n t i m a l g l y c o p r o t e i n was tested f o r i t s LPL i n h i b i t o r y a c t i v i t y by employing post-heparin dog plasma as the source of enzyme and E d i o l ( s t a b i l i z e d coconut o i l emulsion) as the t r i g l y c e r i d e s u b s t r a t e . Because i t i n h i b i t e d post-heparin
Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
203
Lipase
TABLE I C o n c e n t r a t i o n of Component Sugars of Normal and A t h e r o s c l e r o t i c Aortae Component
Sclerotic
Normal
2 Wet weight Xmg/cm ) DDAF (mg/cm ) T o t a l carbohydrate (mg/cm ) Hexose (ug/cm ) Hexosamine (ug/cm ) Uronic a c i d (ug/cm^) S i a l i c a c i d (ug/cm^) T o t a l carbohydrate (ug/mg DDAF) Hexose (ug/mg DDAF) Hexosamine (ug/mg DDAF) Uronic a c i d (ug/mg DDAF) S i a l i c a c i d (ug/mg DDAF)
250.4 (a) 36.9 (b) 2.30 (a) 978 (a) 698 (c) 308 (NS) 317 (b) 63.7 (a) 27.1 (a) 19.4 (a) 8.5 (NS) 8.8 (a)
195.7 39.3 2.10 870 651 297 285 55.4 23.1 17.0 7.9 7.5
2
The c o n c e n t r a t i o n o f components are expressed as weight per u n i t area and weight per u n i t dry, d e f a t t e d , a s h - f r e e r e s i d u e (DDAF). A l l v a l u e s represent averages from 15 samples of aortae obtained from i n d i v i d u a l s of 7 0 . 3 + 2 . 8 (mean + S.E.M.) years of age. P a i r e d t t e s t : (a) = P
