Glycoproteins and Glycolipids in Disease Processes - American

14 Membrane Glycoproteins—Dynamics and Affinity Isolation

Downloaded by EMORY UNIV on August 26, 2015 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0080.ch014

REUBEN LOTAN and GARTH L. NICOLSON Department of Developmental and Cell Biology, University of California, Irvine, CA 92717 Although the exact structure and organization of biological membranes has not been determined in detail, a fair amount of information has been assembled indicating that cellular membranes generally conform to several basic principles. The first of these is that the bulk membrane lipids such as the phospholipids are arranged in a planar bilayer configuration which i s in a predominantly fluid state under physiological conditions (1-4). The lipid bilayer i s interrupted at certain sites by tightly bound integral (or intrinsic) membrane proteins and glycoproteins which are i n serted to varying degrees into the bilayer, and these proteins and glycoproteins are capable of rapid lateral movement within the fluid planar lipid matrix. Cellular membranes are asymmetric with respect to the distribution of specific lipids in each half of the bilayer and proteins and glycoproteins at either surface, and plasma membranes often have non-uniform distributions of proteins, glycoproteins, lipids and glycolipids in the membrane plane (5). In addition, most plasma membranes are not autonomous structures; they are linked to other cellular organelles by a cytoskeletal system composed of microfilaments, microtubules and perhaps intermediate filaments (Fig. 1). However, in its simplest form a biological membrane can be viewed as a two-dimensional solution of a mosaic of integral membrane proteins and glycoproteins embedded in a fluid l i p i d bilayer with peripheral (loosely bound) proteins and glycoproteins attached at the inner and outer membrane surfaces, respectively. The striking asymmetry of this structure allows receptors for hormones, antibodies, viruses, lectins and other agents to be present exclusively on the outer surface where they are exposed to the extracellular environment. Asymmetric arrangement of these components is also well suited to allow the vectorial flow of information across the membrane. The other most important feature of this type of molecular arrangement is that components can diffuse laterally within the membrane plane permitting rapid and reversible changes in membrane topography. Measurements of the lateral mobility of membrane glycoproteins indicate that some diffuse freely in the membrane while others are 0-8412-0452-7/78/47-080-256$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

L O T A N AND NICOLSON

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Glycoproteins

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14.

Cell Surface Reviews

Figure 1. Hypothetical structure of a plasma membrane (PM) including possible interactions between membrane-associated microtubule (MT) and microfilament (MF) systems involved in trans-membrane control over cell surface receptor mobility and distribution (5)



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r e s t r a i n e d and are unable t o undergo r a p i d l a t e r a l movements (4,5_) . This graded h i e r a r c h y o f m o b i l i t i e s f o r d i f f e r e n t components i n the plasma membrane and even w i t h i n c e r t a i n s p e c i f i c regions o f the plasma membrane suggests t h a t a c e l l can c o n t r o l the m o b i l i t i e s o f c e r t a i n membrane components i n order t o maintain c e r t a i n s p e c i f i c topographic arrays o r "patterns" o f c e l l surface recept o r s t h a t are probably important i n i d e n t i f i c a t i o n and a v a r i e t y of other c e l l u l a r phenomena r e q u i r i n g trans-membrane communication. Since these topographic arrangements are dynamic and not s t a t i c , r a p i d and r e v e r s i b l e changes i n membrane topography could occur i n response t o both i n t r a - and e x t r a c e l l u l a r s t i m u l i . MEMBRANE GLYCOPROTEINS The most important c l a s s o f c e l l surface recpetors i n v o l v e d i n trans-membrane receptor processes are the plasma membrane g l y c o p r o t e i n s . Most o f these appear t o be i n t e g r a l components which are i n t e r c a l a t e d i n t o the hydrophobic plasma membrane core t o depths dependent on the amounts o f hydrophobic surface revealed by three-dimensional f o l d i n g o f the molecules i n t o t h e i r " n a t i v e " s t r u c t u r e s . Some o f these i n t e g r a l membrane g l y c o p r o t e i n s span the plasma membrane and have h y d r o p h i l i c regions o f t h e i r s t r u c t u r e s p r o t r u d i n g a t both the e x t r a c e l l u l a r and i n t r a c e l l u l a r membrane surfaces (6-10). A l s o , i t has been found t h a t c e r t a i n membrane g l y c o p r o t e i n s can e x i s t i n o l i g o m e r i c complexes (11-13) o r i n complexes w i t h p e r i p h e r a l membrane p r o t e i n s (14-16). The m a j o r i t y o f c e l l membrane g l y c o p r o t e i n s are i n t e g r a l components s t a b i l i z e d by hydrophobic f o r c e s , and t h e i r s o l u b i l i z a t i o n r e q u i r e s the use o f c h a o t r o p i c agents o r detergents (JL) . Once s o l u b i l i z e d and s t a b i l i z e d i n b u f f e r e d detergent s o l u t i o n s , membrane g l y c o p r o t e i n s can be p u r i f i e d by conventional methods o f g e l f i l t r a t i o n and i o n exchange chromatography ( i n non-ionic detergents) o r by a f f i n i t y chromatography on i n s o l u b i l i z e d l e c t i n s . LECTIN AFFINITY CHROMATOGRAPHY L e c t i n s are p r o t e i n s o r g l y c o p r o t e i n s t h a t b i n d t o mono- and o l i g o s a c c h a r i d e s w i t h remarkable s p e c i f i c i t y (17,18). They usua l l y possess more than one saccharide b i n d i n g s i t e p e r molecule, and t h e i r i n t e r a c t i o n s w i t h carbohydrate-containing polymers resemble antibody-antigen r e a c t i o n s . For example, l e c t i n s can p r e c i p i t a t e p o l y s a c c h a r i d e s and g l y c o p r o t e i n s and a g g l u t i n a t e c e l l s by c r o s s l i n k i n g s a c c h a r i d e - c o n t a i n i n g biopolymers on the surfaces o f adjacent c e l l s . A l a r g e number o f l e c t i n s have been i s o l a t e d and p u r i f i e d , and i n Figure 2 we have l i s t e d some common l e c t i n s a v a i l a b l e t h a t b i n d c e r t a i n o l i g o s a c c h a r i d e s found i n s o l u b l e g l y c o p r o t e i n s (19-23J and c e l l membrane g l y c o p r o t e i n s (2428). Binding o f l e c t i n s t o g l y c o p r o t e i n s o r even c e l l s can be i n h i b i t e d and i n many cases reversed by use o f s p e c i f i c simple sugars o r g l y c o s i d e s , and t h i s i n f o r m a t i o n can be used t o e l u c i date p o s s i b l e carbohydrate determinants on c e l l surface components (Figure 2). However, l e c t i n b i n d i n g t o macromolecules c a r r y i n g h e t e r o - o l i g o s a c c h a r i d e s i d e chains can be i n f l u e n c e d by a v a r i e t y of f a c t o r s which cannot be adequately reproduced i n experiments



14.


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Glycoproteins

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where simple monosaccharide-lectin i n t e r a c t i o n s are measured. Nevertheless, the b i n d i n g o f a l e c t i n i n a s p e c i f i c and r e v e r s i b l e manner t o a macromolecule i s g e n e r a l l y accepted as an i n d i c a t i o n t h a t the molecule i n question contains c e r t a i n carbohydrate sequences, but the unequivocal c h a r a c t e r i z a t i o n o f carbohydrate s e quence i n a l e c t i n receptor r e q u i r e s i t s i s o l a t i o n i n a pure form from the c e l l membrane f o l l o w e d by chemical e l u c i d a t i o n o f i t s structure. Since most membrane g l y c o p r o t e i n s are almost i n s o l u b l e i n n e u t r a l aqueous s o l u t i o n s , p r o t e o l y t i c enzymes have been used t o cleave and remove s o l u b l e surface glycopeptides from c e l l s . Such glycopeptides were then t e s t e d f o r t h e i r a b i l i t y t o i n h i b i t c e l l a g g l u t i n a t i o n , lymphocyte s t i m u l a t i o n o r b i n d i n g o f l e c t i n s t o c e l l s (29-32). The chemical s t r u c t u r e o f a few l e c t i n - r e a c t i v e glycopeptides has been determined (31-32). However, s i n c e the behavior o f membrane g l y c o p r o t e i n s depends not only on t h e i r o l i g o s a c c h a r i d e s i d e chains but a l s o on i n t e r a c t i o n o f the p r o t e i n moiety w i t h other membrane components, s t u d i e s on i n t a c t molecules r a t h e r than i n v e s t i g a t i o n s o f fragmented p o r t i o n s o f the membrane components are r e q u i r e d . For t h i s purpose techniques have been developed t o i s o l a t e and p u r i f y membrane g l y c o p r o t e i n s i n b u f f e r e d detergent s o l u t i o n s by l e c t i n a f f i n i t y chromatography. Using a wide v a r i e t y o f i n s o l u b i l i z e d l e c t i n s , a f f i n i t y chromatography has proven t o be very u s e f u l i n p u r i f y i n g an i n c r e a s i n g number o f membrane g l y c o p r o t e i n s (Table 1 ) . L e c t i n A f f i n i t y P u r i f i c a t i o n o f RBC G l y c o p r o t e i n s The e r y t h r o c y t e membrane has been i n t e n s i v e l y i n v e s t i g a t e d i n an attempt t o understand the s t r u c t u r e and f u n c t i o n o f i t s components (33). As a r e s u l t o f these e f f o r t s the human r e d blood c e l l (HuRBC) membrane has proven t o be a u s e f u l model f o r the development o f methods f o r i s o l a t i o n o f l e c t i n r e c e p t o r s . Indeed, s e v e r a l l a b o r a t o r i e s have succeeded i n p u r i f y i n g HuRBC membrane components to homogeneity using l e c t i n a f f i n i t y chromatography techniques (Table 1 ) . F i n d l a y (34) a p p l i e d s o l u b i l i z e d RBC ghost membrane g l y c o p r o t e i n s onto Con A-Sepharose o r LCA-Sepharose, and a f t e r the unbound m a t e r i a l was removed by washing, the a d d i t i o n o f methyl a-mannoside t o Con A-Sepharose detached a g l y c o p r o t e i n which was i d e n t i f i e d as the major i n t e g r a l g l y c o p r o t e i n o f HuRBC — the component m i g r a t i n g on g e l e l e c t r o p h o r e s i s as Band I I I , a g l y c o p r o t e i n having a mol. wt. o f ^100,000 t h a t contains 5-8% carbohydrate. Use o f the LCA-Sepharose column y i e l d e d the major s i a l o g l y c o p r o t e i n , g l y c o p h o r i n , which contains n e a r l y 60% carbohydrate (24). Band I I I has a l s o been p u r i f i e d on Con A-Sepharose by Ross and McConnell (35) who combined i t w i t h egg l e c i t h i n , e r y t h r o c y t e l i p i d s , c h o l e s t e r o l and g l y c o p h o r i n t o o b t a i n v e s i c l e s which were capable o f s u l f a t e t r a n s p o r t . Two d i s t i n c t c l a s s e s o f HuRBC membrane g l y c o p r o t e i n s w i t h d i f f e r i n g carbohydrate compositions and l e c t i n r e a c t i v i t i e s have been separated by A d a i r and K o r n f e l d (36) using a f f i n i t y chromatography on WGA- and RCAj-Sepharose. The WGA-Sepharose column bound and s p e c i f i c a l l y r e l e a s e d a s i n g l e



f

MW >100 000 IgM, IgD H-2K, H-2D, I a Synaptic g l y c o p r o t e i n s Synaptic g l y c o p r o t e i n s Nicotinic acetylcholine receptor

f

Band I I I Band I I I + g l y c o p h o r i n Band I I I Band I I I + 2 other glycoprote ins Glycophorin Glycophorin A A s i a l o g l y c o p h o r i n (T-Ag) Major g l y c o p r o t e i n Glycoproteins Glycoprote i n s HLA antigens Component 5.1 T-25, T-200 Major antigen H-2K, H-2D, I a IgM IgD Glycoproteins: MW 20,000-80,000

Material Purified

Table 1:

Mu lymphocytes Mu B lymphocytes Mu lymphocytes Rat b r a i n Rat b r a i n Rat b r a i n o r e l e c t r i c e e l

TX-100 TX-100 TX-100 NaDOC SDS TX-100

PA-^CA^CAj48 Seph WGA, BPA-Seph 48 PA-Seph 48 PA-Seph 48 LCA-, WGA-Seph 49 Con A-, UEA-Seph 50,51 Con A, WGA-Seph, 52 RCAj-glass beads

TX-100

Mu lymphocytes

36 37 38 40 41 42 43 44 45 46 47 47

36

RCAj-Seph WGA-Seph WGA-Seph PNA-Seph RCAj-Seph Con A-Seph LCA-Seph LCA-Seph Con A-Seph Con A, PA-Seph LCA-Seph Con A-Seph Con A-Seph

TX-100 TX-100 SDS DDG (Empigen BB) Emulphogene NaDOC NaDOC NaDOC TX-100 NP-40, NaDOC NaDOC NP-40 NP-40

34 34 35

Ref.

Con A-Seph LCA-Seph Con A-Seph

Affinity Adsorbent

HuRBC

TX-100 TX-100 DTAB

Membrane S o l u b i l i z i n g Agent

HuRBC HuRBC HuRBC + NANase Bovine RBC P i g lymphocytes P i g lymphocytes Hu lymphocytes Ra thymocytes Mu thymocytes Rat thymocytes Mu thymocytes Mu B lymphocytes

HuRBC HuRBC HuRBC

Source o f Membrane

ISOLATION AND CHARACTERIZATION OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS



Rat spermatozoa Enveloped v i r u s e s ( i n f l u e n z a , Sendai) Mu mammary tumor v i r u s D i c t y o s t e l i u m discoidum

Con A-receptors V i r a l glycoprotein

TX-100 NaDOC

Membrane S o l u b i l i z i n g Agent NaDOC CTAB LIS LIS LIS SDS DDG NaDOC

Affinity Adsorbent Con A-Seph Con A-Seph Con A-Seph WGA-Seph Con A-Seph WGA-Seph RCAj-Seph RCAj-, Con A-, WGA, SBA-Seph Con A-Seph LCA-Seph 61 62

Ref. 53 54 55 56 57 58 59 60

Serdox NNP10 Con A-Seph Major g l y c o p e p t i d e s 63 Membrane-bound enzymes NaDOC Con A-Seph 64 (cf, 5'-nucleotidase, a l k a l i n e phosphatase) A b b r e v i a t i o n s : BPA, Bauhinia purpurea a g g l u t i n i n ; CTAB, cetyltrimethylammonium bromide; DDG, d i m e t h y l dodecylglycine (Empigen BB); DTAB, dodecyltrimethy1ammonium bromide; Emulphogene, a l k o x y p o l y (ethyleneoxy)ethanol; Ham, hamster; Hu, human; HuRBC, human r e d blood c e l l s ; Mu, murine; NaDOC, sodium deoxycholate; NP-40, Nonidet P-40, p o l y o x y e t h y l e n e g l y c o l ( 6 - 7 ) - p - t - o c t y l p h e n o l ; Ra, r a b b i t ; SDS, sodium dodecyl s u l f a t e ; Seph, Sepharose; Serdox NNP10, a n o n i o n i c detergent, Servo, Delden, The Netherlands; TX-100, T r i t o n X-100, p o l y o x y e t h y l e n e g l y c o l ( 9 - 1 0 ) - p - t - o c t y l p h e n o l .

Source o f Membrane Bovine b r a i n Bovine r e t i n a Hu p l a t e l e t s Mu LI210 lymphoma Mu L-929 c e l l s Hu HeLa c e l l s Ham embryo f i b r o b l a s t s (NIL) Mu E h r l i c h a s c i t e s carcinoma

OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS

Material Purified Tissue f a c t o r Rhodopsin Major g l y c o p r o t e i n WGA-receptors Con A-receptor WGA-receptors G a l a c t o p r o t e i n s a and b Glycoproteins

Table 1 ( c o n ' t . ) : ISOLATION AND CHARACTERIZATION


GLYCOPROTEINS AND

262

GLYCOLIPIDS IN

DISEASE PROCESSES

g l y c o p r o t e i n (glycophorin) i n h i b i t o r of WGA, Agaricus bisporus a g g l u t i n i n and PHA. The RCAj-Sepharose column adsorbed s e v e r a l g l y c p r o t e i n s c o n t a i n i n g b i n d i n g s i t e s f o r RCAj and Abrus p r e c a t o r i u s a g g l u t i n i n , while most of the g l y c o p r o t e i n receptors f o r A. bisporus a g g l u t i n i n , PHA, LCA and WGA were not r e t a i n e d by the column. P u r i f i c a t i o n of g l y c o p h o r i n i n a one-step p r e p a r a t i v e procedure was obtained by Kahane et a l . (37) a f t e r SDS s o l u b i l i z a t i o n of HuRBC ghosts i n the presence of r e l a t i v e l y high s a l t and a d s o r p t i o n to WGA-Sepharose. The r e c e p t o r f o r peanut a g g l u t i n i n was i s o l a t e d from neuraminidase-treated, dimethyl d o d e c y l g l y c i n e s o l u b i l i z e d HuRBC by chromatography on PNA-polyacrylhydrazidoSepharose (38). In t h i s l a s t procedure c e l l surface galactose residues on i n t a c t neuraminidase-treated HuRBC were r a d i o l a b e l e d by o x i d a t i o n w i t h galactose oxidase f o l l o w e d by r e d u c t i o n w i t h N a B H t p r i o r to t h e i r p u r i f i c a t i o n . The use of p o l y a c r y l h y d r a zido-Sepharose a f f o r d e d a high c a p a c i t y , charge-free and nonl e a c h i n g supporting m a t r i x which e x h i b i t e d very low n o n s p e c i f i c a d s o r p t i o n and high r e c o v e r i e s (38,39). The m a t e r i a l e l u t e d from PNA-polyacrylhydrazido-Sepharose w i t h l a c t o s e c o n s i s t e d of two g l y c o p r o t e i n s (MW ^82,000 and ^46,000, r e s p e c t i v e l y ) which resemb l e d a s i a l o g l y c o p h o r i n i n e l e c t r o p h o r e t i c m i g r a t i o n and amino a c i d composition. L i k e a s i a l o g l y c o p h o r i n , the PNA receptor formed p r e c i p i t i n bands w i t h SBA and RCA-j- but f a i l e d to p r e c i p i t a t e w i t h Con A, Dolichos b i f l o r u s a g g l u t i n i n and WGA (38). In an e f f o r t to determine the degree to which the carbohydrate s i d e chains of HuRBC membrane g l y c o p r o t e i n s vary between s p e c i e s , Emerson and K o r n f e l d (40) used non-ionic detergent s o l u b i l i z e d bovine e r y t h r o c y t e membranes as a source of g l y c o p r o t e i n s . RCAjSepharose adsorbed the 230,000 mol. wt. major g l y c o p r o t e i n of bovine e r y t h r o c y t e membrane which contains ^80% carbohydrate, mostly l i n k e d o - g l y c o s i d i c a l l y . V i r t u a l l y a l l the carbohydrate of the membrane (excluding g l y c o l i p i d s ) was borne by t h i s g l y c o p r o t e i n . L e c t i n A f f i n i t y P u r i f i c a t i o n of Lymphoid C e l l Membrane Components Lymphoid c e l l membranes c o n t a i n a wide v a r i e t y of g l y c o p r o t e i n molecules compared to RBC; t h e r e f o r e , l e c t i n a f f i n i t y columns are g e n e r a l l y u s e f u l f o r p a r t i a l p u r i f i c a t i o n or enrichment of g l y c o p r o t e i n s . For example, almost a l l the g l y c o p r o t e i n s of p i g lymphocyte plasma membrane s o l u b i l i z e d i n sodium deoxycholate (NaDOC) were adsorbed on columns of Con A-Sepharose (41) or LCA-Sepharose (42). The l a t t e r immobilized l e c t i n was much more e f f i c i e n t i n p u r i f y i n g the s o l u b i l i z e d membrane g l y c o p r o t e i n s . Con A-Sepharose bound 20% of the a p p l i e d m a t e r i a l i n a n o n s p e c i f i c manner, and the y i e l d of g l y c o p r o t e i n s o f f the Con A-Sepharose column was low ( 5 % ) , whereas LCA-Sepharose y i e l d e d more than twice the amount obtained w i t h Con A-Sepharose, and the recovery was 95%. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) of the s a c c h a r i d e - e l u t e d LCA-Sepharose f r a c t i o n s revealed a t l e a s t ten p r o t e i n bands t h a t s t a i n e d f o r carbohydrate w i t h p e r i o d a t e - S c h i f f reagent. P u r i f i e d LCAreceptors i n h i b i t e d LCA-induced lymphocyte t r a n s f o r m a t i o n 10 times


3

+



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Glycoproteins

263

more e f f e c t i v e l y compared t o the u n f r a c t i o n a t e d membrane g l y c o p r o t e i n s , and a t l e a s t one o f the LCA-receptor g l y c o p r o t e i n s was a l s o capable o f i n h i b i t i n g PHA-induced lymphocyte s t i m u l a t i o n . I n the above s t u d i e s f u r t h e r c h a r a c t e r i z a t i o n was not performed t o e l u c i d a t e the nature and f u n c t i o n o f the l e c t i n r e a c t i v e m a t e r i a l s ; however, i n other r e p o r t s defined components have been i s o l a t e d by l e c t i n a f f i n i t y chromatography. For example, the h i s t o c o m p a t i b i l i t y antigens o f human lymphocytes (HLA) were s o l u b i l i z e d i n sodium deoxycholate and then p u r i f i e d by chromatography on LCA-Sepharose (43) . Improvements have been made i n l e c t i n a f f i n i t y chromatography techniques t o increase s p e c i f i c i t y and y i e l d as w e l l as reduce nons p e c i f i c a d s o r p t i o n . In an attempt t o increase the s e l e c t i v i t y o f lectin-Sepharose chromatography, S c h m i d t - U l l r i c h e t a l . (4£) adsorbed T r i t o n X - 1 0 0 - s o l u b i l i z e d r a b b i t thymocyte membrane g l y c o p r o t e i n s onto Con A-Sepharose as i n other procedures. However, the e l u t i o n was performed i n a stepwise f a s h i o n using i n c r e a s i n g conc e n t r a t i o n s o f the hapten methyl a-glucoside. This procedure a l lowed p r e f e r e n t i a l e l u t i o n o f receptors according t o t h e i r r e l a t i v e b i n d i n g a f f i n i t i e s t o Con A. Using these procedures a s i a l o g l y c o p r o t e i n t h a t has been designated component 5.1 because o f i t s e l e c t r o p h o r e t i c m o b i l i t y i n SDS-PAGE was e l u t e d i n a homogeneous form. This Con A receptor has an apparent MW o f 55,000 d a l t o n s but tends t o aggregate i n s o l u t i o n . When t h i s p r e p a r a t i o n was examined f u r t h e r , two molecules were found t h a t d i f f e r i n t h e i r carbohydrate side chains, and these could be recognized by cross immune e l e c t r o p h o r e s i s . Trowbridge e t a l . (45) reported t h a t specific I - l a b e l e d surface antigens (T25 and T200) on mouse thymocytes could be p u r i f i e d from NaDOC-solubilized membranes by a d s o r p t i o n t o e i t h e r Con A- o r PA-Sepharose. Upon a n a l y s i s by SDS-PAGE these components separated i n t o s e v e r a l s p e c i e s , p o s s i b l y due to heterogeneity o f t h e i r carbohydrate s i d e chains. Fabre and W i l l i a m s (46) i s o l a t e d a major antigen from the surfaces o f r a t thymocyte membranes by s p e c i f i c a d s o r p t i o n on LCA-Sepharose, and t h i s behavior demonstrated t h a t the antigen i s a g l y c o p r o t e i n . N i l s s o n and Waxdal (47) a l s o l a b e l e d p r o t e i n s on the surface o f murine lymphocytes by l a c t o p e r o x i d a s e - c a t a l y z e d I - i o d i n a t i o n or by c u l t u r i n g the c e l l s i n media c o n t a i n i n g H - l e u c i n e o r H fucose. C e l l membranes were s o l u b i l i z e d i n NP-40 before p a s s i n g the s o l u b i l i z e d g l y c o p r o t e i n s through Con A-Sepharose. The m a t e r i a l s p e c i f i c a l l y e l u t e d from Con A-Sepharose was c h a r a c t e r i z e d by imm u n o p r e c i p i t a t i o n w i t h s p e c i f i c a n t i s e r a , and i t was found t h a t Con A-receptors i n c l u d e d antigens coded by the h i s t o c o m p a t i b i l i t y - 2 complex such as H-2K, H-2D and l a . I n a d d i t i o n , the immobilized Con A a l s o adsorbed immunoglobulins M and D from s o l u b i l i z e d B lymphocyte membranes. E s s e n t i a l l y the same r e s u l t s were obtained r e c e n t l y by Iwata e t a l . (48) who e x t e n s i v e l y f r a c t i o n a t e d murine lymphoid c e l l surface components on a b a t t e r y o f lectin-Sepharose columns (see Table 1). This l a s t study demonstrated t h a t the use of s e v e r a l lectin-Sepharose columns f o r f r a c t i o n a t i o n o f c e l l 1 2 5

1 2 5

3


3

GLYCOPROTEINS AND

264

GLYCOLIPIDS IN

DISEASE PROCESSES

membrane components narrows the range of i s o l a t e d components. For example, PLA-, LCA- and RCAj-Sepharose s p e c i f i c a l l y bound g l y c o p r o t e i n s of MW i n the range 20,000-80,000 d a l t o n s , whereas g l y c o p r o t e i n s i n the higher MW (>100,000) range t h a t were not adsorbed on the l a t t e r columns were p r e f e r e n t i a l l y bound by WGA- and BPASepharose (48). L e c t i n A f f i n i t y Chromatography of Nerve C e l l Components L e c t i n a f f i n i t y chromatography has been e s p e c i a l l y u s e f u l i n the p u r i f i c a t i o n of carbohydrate-containing macromolecules from nervous t i s s u e . G l y c o p r o t e i n s present i n NaDOC-solubilized syna p t i c plasma membranes prepared from r a t b r a i n s t h a t were i n j e c t e d w i t h H-fucose 16 hr e a r l i e r have been f r a c t i o n a t e d by chromatography on LCA- or WGA-Sepharose (49). In these s t u d i e s LCA-Sepharose adsorbed 40-45% of the a p p l i e d r a d i o a c t i v i t y and bound most of the 7-8 g l y c o p r o t e i n s known to be present i n the s y n a p t i c membranes; whereas WGA-Sepharose bound o n l y 25-30% of the r a d i o a c t i v i t y suggesting t h a t some of the g l y c o p r o t e i n s r e t a i n e d on the LCA-Sepharose were not bound by the WGA column. Therefore, Gurd and Mahler (49) decided t o use both of these a f f i n i t y columns i n tandem such t h a t the sample a p p l i e d onto LCA-Sepharose was allowed to pass i n t o the WGA-Sepharose column. A f t e r washing both columns and s e q u e n t i a l l y e l u t i n g w i t h the appropriate monosaccharide i n h i b i t o r s f o r each l e c t i n , four f r a c t i o n s were obtained: LCA-negative, WGA-negative; LCA-negative, WGA-positive; L C A - p o s i t i v e , WGA-negat i v e ; and L C A - p o s i t i v e , WGA-positive. When analyzed by SDS-PAGE, each of the f r a c t i o n s had d i s t i n c t p a t t e r n s w i t h some o v e r l a p of molecular weight c l a s s e s . Zanetta e t a l . (50,51) have f r a c t i o n a t e d r a t b r a i n s y n a p t i c membranes d i s s o l v e d i n SDS on Con A- or UEASepharose. E l u t i o n from the former a f f i n i t y column was w i t h methyl a-glucoside; however, L-fucose was unable to r e l e a s e bound m a t e r i a l but c o u l d i n h i b i t b i n d i n g t o the UEA-Sepharose. I n c r e a s i n g the SDS c o n c e n t r a t i o n i n the e l u t i o n b u f f e r (without saccharides) r e leased the adsorbed m a t e r i a l , and subsequent PAGE a n a l y s i s revealed a l a r g e number of carbohydrate c o n t a i n i n g components. P a r t of the heterogeneity i n g l y c o p r o t e i n s e l u t e d by these procedures was a t t r i b u t e d t o the f a c t t h a t the s y n a p t i c membranes were d e r i v e d from a heterogeneous p o p u l a t i o n of neurons. S p e c i f i c , f u n c t i o n a l g l y c o p r o t e i n s have been i s o l a t e d from s o l u b i l i z e d b r a i n t i s s u e by lectin-Sepharose chromatography. Nicot i n i c a c e t y l c h o l i n e r e c e p t o r , T r i t o n X-100 s o l u b i l i z e d from a crude, p o s t - n u c l e a r membrane f r a c t i o n of r a t c e r e b r a l c o r t e x or from membranes of Torpedo c a l i f o r n i c a e l e c t r o p l a x , was r e t a i n e d on immobilized Con A, WGA or RCAj (52) . In another study p a r t i a l p u r i f i c a t i o n of coagulant arylamidase and a l k a l i n e phosphatase enzymes s o l u b i l i z e d i n NaDOC from bovine b r a i n was achieved by a d s o r p t i o n on Con A-Sepharose (53). L e c t i n A f f i n i t y Chromatography of Components from Other C e l l Types In c o n t r a s t to the m u l t i t u d e of l e c t i n receptors found i n s y n a p t i c membranes, s e v e r a l systems have been described i n which


3



14.

Membrane

Glycoproteins

265

a s i n g l e receptor was obtained. For example, s o l u b i l i z a t i o n o f bovine r e t i n a l r o d outer segments w i t h cetyltrimethylammonium bromide f o l l o w e d by chromatography on Con A-Sepharose y i e l d e d a homogeneous rhodopsin (54). The major human p l a t e l e t membrane g l y c o p r o t e i n (MW ^100,000) was s o l u b i l i z e d by l i t h i u m d i i o d o s a l i c y l a t e -phenol e x t r a c t i o n o f I - l a b e l e d membranes and p u r i f i e d on Con ASepharose (55). Using s i m i l a r e x t r a c t i o n methods, four WGA-recept o r s o f MW 40,000-60,000 were i s o l a t e d from L1210 c e l l s (560, and a s i n g l e Con A receptor having an apparent MW o f 100,000 and a t l e a s t f i v e carbohydrate s i d e chains was i s o l a t e d from mouse L929 c e l l s (57J. M u l t i p l e WGA receptors o f MW i n the range 40,000300,000 were i s o l a t e d from S D S - s o l u b i l i z e d membranes o f il a b e l e d HeLa c e l l s (58). E x t r a c t i o n o f hamster embryo f i b r o b l a s t s (NIL) l a b e l e d w i t h galactose-oxidase and NaB^H^ w i t h 8M urea o r s o l u b i l i z a t i o n w i t h d i e m t h y l d o d e c y l g l y c i n e f o l l o w e d by chromatography on RCASepharose allowed p u r i f i c a t i o n o f g a l a c t o p r o t e i n a (LETS) and g a l a c t o p r o t e i n b (59J . G a l a c t o p r o t e i n a was accompanied by an a c t i n - l i k e p r o t e i n , and g a l a c t o p r o t e i n b appeared composit i o n a l l y heterogeneous. While most o f the above i n v e s t i g a t o r s d i d not r e p o r t problems i n u s i n g lectin-Sepharose columns, an e x t e n s i v e study by Nachbar e t a l . (60) demonstrated t h a t i n c e l l membranes o f E h r l i c h a s c i t e s carcinoma c e l l s s o l u b i l i z e d i n NaDOC there are s e v e r a l carbohydrate c o n t a i n i n g components t h a t adsorb t o Con A-, WGA-, SBA- and RCA-Sepharose columns. Using C-glucosaminel a b e l e d membrane components the r e c o v e r i e s from the v a r i o u s columns were determined, and i t was found t h a t s p e c i f i c e l u t i o n o f the WGAand the Con A-Sepharose columns r e s u l t e d i n r e l e a s e o f only 55-70% of the bound m a t e r i a l . The remaining t i g h t l y bound m a t e r i a l may have been i r r e v e r s i b l y h e l d t o the column by hydrophobic i n t e r a c t i o n s . The r e c e p t o r s f o r the d i f f e r e n t l e c t i n s appeared s i m i l a r on SDS-PAGE, and some immobilized l e c t i n s p u r i f i e d the same g l y c o p r o t e i n r e c e p t o r s . P u r i f i c a t i o n o f g l y c o p r o t e i n s from s o l u b i l i z e d enveloped v i r u s e s (62,63) and o f membrane-associated enzymes on l e c t i n a f f i n i t y columns has a l s o been d e s c r i b e d (Table 1 ) . Although one-step p u r i f i c a t i o n t o homogeneity o f a membrane g l y c o p r o t e i n has only been reported f o r HuRBC g l y c o p h o r i n (37), most s t u d i e s i n d i c a t e t h a t a f f i n i t y chromatography on immobilized l e c t i n columns i s the method o f c h o i c e , a t l e a s t i n p r e l i m i n a r y stages o f membrane g l y c o p r o t e i n p u r i f i c a t i o n . In e a r l i e r s t u d i e s (34,36,41,42) no attempt was made t o r a d i o a c t i v e l y l a b e l c e l l membrane components p r i o r t o s o l u b i l i z a t i o n and chromatography, b u t more recent i n v e s t i g a t i o n s have demonstrated t h a t p r e l a b e l i n g membrane components e i t h e r by l a c t o p e r o x i d a s e - c a t a l y z e d I-iodinat i o n (47,55) o r neuraminidase f o l l o w e d by galactose oxidase and NaB Hi (_38/57) f a c i l i t a t e s the f r a c t i o n a t i o n procedure and i n s u r e s t h a t the i s o l a t e d components o r i g i n a t e from the plasma membrane s u r f a c e . I n a d d i t i o n , the l a b e l e d components may be detected by autoradiography o r fluorography a f t e r s e p a r a t i o n by SDS-PAGE, even i f they are obtained i n s m a l l q u a n t i t i e s . Many o f the saccharides found i n membrane g l y c o p r o t e i n s are 1 2 5


1 2 5

ll+

1 2 5

3

+



266


u b i q u i t o u s ; t h e r e f o r e , o n l y p a r t i a l p u r i f i c a t i o n should be expected when a s i n g l e lectin-Sepharose column i s used. The use o f a hapten g r a d i e n t i n the e l u t i o n step (44) o r combinations o f immobilized l e c t i n s w i t h d i f f e r e n t sugar s p e c i f i c i t i e s (48,49) has proven t o be very powerful i n the s e p a r a t i o n o f membrane g l y c o p r o t e i n s . The choice o f detergent f o r the s o l u b i l i z a t i o n o f membranes and s t a b i l i z a t i o n o f membrane g l y c o p r o t e i n s p r i o r t o a p p l i c a t i o n onto lectin-Sepharose columns seems i n many r e p o r t s t o have been random, or i n the best examples, e m p i r i c a l . This problem has been e l i m i nated i n p a r t by a systematic a n a l y s i s o f the e f f e c t s o f s e v e r a l z w i t t e r i o n i c , c a t i o n i c , a n i o n i c and non-ionic detergents on g l y c o p r o t e i n b i n d i n g t o and s p e c i f i c e l u t i o n from f i v e lectin-Sepharose columns (39). Our r e s u l t s (Figure 3) i n d i c a t e t h a t f o r most l e c t i n s , NaDOC caused marked decreases i n b i n d i n g a f f i n i t y . The z w i t t e r i o n i c detergent dimethyl d o d e c y l g l y c i n e decreased the e f f i c i e n c y o f the Con A- and SBA-Sepharose, w h i l e the non-ionic detergents NP-40 and T r i t o n X-100 were s u i t a b l e f o r a l l the immob i l i z e d l e c t i n s t e s t e d (39_) . Future use o f s p e c i f i c l e c t i n a f f i n i t y chromatography techniques w i l l be g r e a t l y f a c i l i t a t e d by opt i m i z i n g v a r i o u s l e c t i n s , detergents, s o l u t i o n c o n d i t i o n s , columns and techniques f o r s p e c i f i c e l u t i o n o f bound g l y c o p r o t e i n s . DYNAMICS OF CELL SURFACE LECTIN RECEPTORS One o f the most u s e f u l approaches f o r studying c e l l s u r f a c e g l y c o p r o t e i n dynamics has been the u t i l i z a t i o n o f l e c t i n s as probes f o r f l u o r e s c e n t o r e l e c t r o n microscopy (18,65). Observing the movement and r e d i s t r i b u t i o n o f c e l l surface-bound l e c t i n s has allowed estimates t o be made on the r a t e s o f l a t e r a l d i f f u s i o n and, i n c e r t a i n cases, i n t e r n a l i z a t i o n o f the l e c t i n - r e c e p t o r comp l e x e s (_5) . These experiments have been g e n e r a l l y conducted w i t h f l u o r e s c e i n - l a b e l e d l e c t i n s and UV-fluorescent microscopy o r f e r r i t i n - , hemocyanin- o r p e r o x i d a s e - l a b e l e d l e c t i n s and e l e c t r o n microscopy (65). By l a b e l i n g p r e f i x e d o r u n f i x e d c e l l s u r f a c e s w i t h these probes and then observing the a l t e r a t i o n s i n s u r f a c e d i s t r i b u t i o n s o f the probes under a v a r i e t y o f experimental c o n d i t i o n s , a dynamic p i c t u r e o f l e c t i n r e c e p t o r movements has been obtained i n s e v e r a l b i o l o g i c a l systems. These observations have l e d i n v e s t i g a t o r s t o conclude the f o l l o w i n g : F i r s t , the i n h e r e n t topographic d i s t r i b u t i o n s o f most l e c t i n r e c e p t o r s (as found on p r e f i x e d c e l l s o r c e l l s l a b e l e d a t 0°C) are uniform and random across the e n t i r e c e l l s u r f a c e . Some exceptions have been noted, however, i n s p e c i a l i z e d c e l l s o f h i g h asymmetry such as sperm where WGA-receptors were found t o be l o c a l i z e d o n l y i n c e r t a i n regions o f the sperm head (66) and nerve c e l l s where Con A recept o r s were found t o be r e l a t i v e l y immobile and predominately i n the s y n a p t i c c l e f t (67). Most l e c t i n r e c e p t o r r e d i s t r i b u t i o n s t u d i e s have r e v e a l e d t h a t l i g a n d - r e c e p t o r complexes undergo r a p i d change from random, uniform d i s t r i b u t i o n s t o form c l u s t e r s and patches (68-72). I n some c e l l types the c l u s t e r e d l i g a n d - r e c e p t o r complexes coalesce t o form a s i n g l e p o l a r "cap" o r aggregate o f c r o s s l i n k e d l i g a n d - r e c e p t o r complexes. A f t e r r e d i s t r i b u t i o n the c l u s -


14.


Membrane

Glycoproteins

I. Serum Glycoprotein-Type

LECTIN

267

Oligosaccharide

NANA-Gal-GlcNAc-Man. Man-GlcNAc-GlcNAc-Asn

LPA Gal-GlcNAc-Man*

t Fuc

RCA,SBA,PNA PHA WGA Con A,LCA,PA LTA,UEA


I I . Mucin-Type

Oligosaccharide NANA-Gal-GalNAc-Ser(Thr)

LPA

NANA Gal-GalNAc-Ser(Thr)

PNA

I

1

SBA,RCA Figure 2. Possible binding region for lectins on oligosaccharide side chains of a hypothetical cell membrane glycoprotein. LPA, Limulus polyphemus agglutinin; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; PNA, peanut agglutinin; PHA, phytohemagglutinin from Phaseolus vulgaris; WGA, wheat germ agglutinin; Con A, concanavalin A; LCA, Lens culinaris agglutinin; PA, pea agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europeus agglutinin.

• RC

60 20

1 t 1 I it 1 J I 11 TXIOOPj,

100 60 20

3

100 60 20 100

a SBA • Con Am WGA

PNA

NP40

100

-D

DDG

1 m 1 ii y DOC

1

60 20 100 60 20

•E

c

i! i 2

0.1

T A B

1.0

DETERGENT CONC. (%)

2.5

Biochemistry

Figure 3. Effect of increasing concentrations of detergents on the glycoprotein binding efficiencies of immobilized lectins (for experimental details see Ref. 39)



268

GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES

t e r e d o r capped l i g a n d - r e c e p t o r complexes may be i n t e r n a l i z e d by endocytosis o r shed from the c e l l . The extents and r a t e s o f r e d i s t r i b u t i o n , i n t e r n a l i z a t i o n and shedding appear t o be c o n t r o l l e d by the c e l l type and the biochemical nature o f the l i g a n d and i t s r e c e p t o r s (reviewed i n 4,18,73). I t has been noted t h a t w h i l e a l e c t i n a t one c o n c e n t r a t i o n can induce r e d i s t r i b u t i o n o f i t s r e ceptors i n t o p o l a r caps, a t other c o n c e n t r a t i o n s ( u s u a l l y much h i g h e r ) , i t can i n h i b i t the movement o f d i f f e r e n t c e l l surface r e c e p t o r s (74,751 Q u a n t i t a t i o n o f the r o t a t i o n a l and l a t e r a l m o b i l i t i e s o f c e l l surface g l y c o p r o t e i n s on c e l l surfaces has been achieved by use o f f l u o r e s c e i n i s o t h i o c y a n a t e - l a b e l e d Con A. S h i n i t z k y e t a l . (76) and Inbar e t a l . (77) have s t u d i e d the r o t a t i o n a l m o b i l i t i e s o f Con A receptor complexes on c e l l surfaces by fluorescence p o l a r i z a t i o n . The recent i n t r o d u c t i o n o f the fluorescence photobleaching recovelry technique allowed Jacobson e t a l . (78) and S c h l e s s i n g e r e t a l (79) t o measure l a t e r a l d i f f u s i o n c o e f f i c i e n t s f o r the Con A-receptor complexes w i t h i n s u r f a c e membranes o f s e v e r a l c u l t u r e d c e l l s . Such s t u d i e s and others i n d i c a t e d t h a t the m o b i l i t i e s o f c e l l membrane g l y c o p r o t e i n s are determined t o a l a r g e ext e n t by t h e i r i n t e r a c t i o n s w i t h other membrane components and membrane-associated s t r u c t u r e s such as m i c r o f i l a m e n t s and microtubules (4,5^) . I t i s hoped t h a t i s o l a t i o n and c h a r a c t e r i z a t i o n of s p e c i f i c membrane g l y c o p r o t e i n s w i l l increase the understanding of the exact molecular s t r u c t u r e and o r g a n i z a t i o n o f c e l l membranes. Acknowledgments; Supported by PHS NCI Contract CB-74153, PHS NCI Grant CA-15122 and American Cancer S o c i e t y Grant BC-211A t o G.L. Nicolson.


14.


Membrane

Glycoproteins

269

LITERATURE CITED

1. 2. 3. 4. 5.

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Glycoproteins and Glycolipids in Disease Processes - American

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