Molecular flypaper and atherosclerosis: structure of the macrophage scavenger receptor
THE INTERCF.I.LULAR TRANSPORT of lipids, including cholesterol, choles- teryl esters, triglycerides and certain vitamins and drugs, through the aqueous circulatory system requires that these hydrophobic molecules be packaged into water-soluble carriers called lipo- proteins. There are several different classes of mammalian lipoproteins, most of which have been named based on their differing buoyant densities, e.g. low density lipoprotein (LDL), high den- sity lipoprotein (HDL) and very low density lipoprotein (VLDL)L The lipo- proteins differ in their protein and lipid compositions, their sizes and their physiological and pathophysiological activities. For example, there is a direct correlation of risk for coronary artery disease with plasma LDL concentration and an inverse correlation with plasma HDL concentration z,3. A model of the structure of LDL, the principle choles- terol transporter in human plasma, is shown in Fig. 1. Two pathways for the cellular uptake of plasma LDL have been the subjects of intense investi- gation: the LDL receptor pathway and the scavenger receptor pathway. The LDL receptor pathway of endocytosis was discovered and has been charac- terized in great detail by Brown, Goldstein and their colleagues (see Ref. 3 for a recent review). By contrast, the cell and molecular biology and the physiological significance of the scaven- ger receptor pathway are much less well defined. However, studies over the past decade have suggested that the scavenger receptor pathway may play a key role in the deposition of lipoprotein cholesterol in artery walls during the formation of atherosclerotic plaques 2,4 and may also be involved in certain host defense mechanisms against patho- gen~ organisms S,6.
Atherosclerosis and the macrophage scavenger receptor model
The hallmark of atherosclerosis is the formation of atherosclerotic plaques in artery walls. A plaque results from a build-up underneath the artery's endo- thelial lining of cells, especially of smooth muscle cells and macrophages, cell debris, extracellular matrix, and
Macrophage scavenger receptors have been implicated both in the depo- sition of lipoprotein cholesterol in artery walls during the formation of atherosclerotic plaques and in host defense against pathogenic infec- tions. The receptor's unusual ability to bind tightly to a very wide variety of ligands and its novel mosaic structure comprising (z-helical coiled-coil, collagenous and cysteine-rich domains are described.
cholesterol and cholesteryl esters. The macrophages and smooth muscle cells in plaques are often filled with chol- esteryl ester-rich foam-like fat droplets which give these foam cells a dis- tinctive microscopic appearance. By protruding into the lumen of a coronary artery, a plaque can narrow the lumen and thus reduce blood flow (one cause of angina) and increase the likelihood of blockage by a blood clot (a major cause of heart attacks).
It is generally agreed that athero- sclerotic plaque formation begins with the attachment of monocytes to the
Phc Cholesterol
Figure 1 Structure of LDL. LDL is a large particle (~2.5 x 106 Da) consisting of a hydro- phobic core containing about 1500 chol- esteryl ester molecules and an amphi- pathic shell of unesterified cholesterol, phospholipid and apolipoprotein B (~513 kDa). Apolipoprotein B is responsible for the binding of native LDL to LDL re- ceptors and of chemically modified LDL to scavenger receptors.
lumenal surface of the endothelium and their subsequent migration into the subendothelial space. There the mono- cytes differentiate into macrophages, and, if plasma LDL levels are high, they accumulate massive amounts of lipo- protein cholesterol and become foam cells 4. Several lines of evidence suggest that the LDL receptor pathway is not required for and may not normally be involved in cholesterol accumulation during foam cell development. For example, in humans and animals with genetic defects in LDL receptor func- tion, plasma LDL levels are abnormally high and plaque formation is acceler- ated 3. Currently, an attractive model for lipoprotein-cholesterol accumulation in macrophage foam cells is the macro- phage scavenger receptor modeF -4. Macrophages, but not their precursor monocytes, express high levels of a receptor activity, discovered by Brown, Goldstein and colleagues 2, which can mediate the endocytosis of chemically modified LDL and thus the conversion of cultured macrophages into foam-like cells. The receptor, which was originally referred to as the acetyl LDL (AcLDL) receptor, is now called the macrophage scavenger receptor because of its dis- tinctive ligand-binding properties (see below). Effective chemical modifi- cations which convert LDL into a ligand of the scavenger receptor include acetyl- ation or oxidation. The identification of oxidized LDL (OxLDL) in plaques, the discovery that the antioxidant probucol can inhibit plaque formation in an ani- mal model of atherosclerosis (for review see Ref. 4), and the ability of scavenger
Figure 2 Two- (a) and three-stranded (b) c(-helical coiled-coils. The seven-amino acid heptad repeat (a-g) in which the a and d positions frequently have aliphatic side chains (leucine, isoleucine, valine) folds into two turns of right-handed c¢-helix. Two or three strands of such helices assemble into parallel left-handed supercoiled bundles which are held together, in part, by a hydrophobic core of the residues in the a and d positions. Examples of proteins containing two- (leucine zipper) or three-stranded c(-helical coiled-coils are listed (see Ref. 19).
receptors to mediate macrophage-foam cell formation in vitro have focused attention on the properties of scav- enger receptors and their potential role in atherogenesis.
Broad ligand-binding specificity One of the most distinctive features
of scavenger receptors is their broad ligand-binding specificity 2. Typical cell surface receptors, such as the insulin or LDL receptors, bind their ligands with high affinity (nanomolar or lower
dissociation constants) and very high or narrow specificity. Macrophage scav- enger receptors also bind ligands with high affinity; however, they bind an extraordinarily wide variety of ligands. High-affinity ligands include some chemically modified proteins (AcLDL, OxLDL, maleylated bovine serum albu- min), certain polyribonucleotides [e.g. poly(I)], some polysaccharides (e.g. carragheenan), certain phospholipids TM
(e.g. phosphatidylserine), polyvinyl sul- fate and bacterial lipopolysaccharide 8,9.
Table I. Proteins containing scavenger receptor cysteine-rich (SRCR) domains
Protein a
Number of SRCR SRCR domains domain
per chain Source Protein function function
Scavenger receptor, 1 Mammalian Binding and type I macrophages endocytosis of
diverse ligands
CD5 (LY-1)
Unknown
3 Mammalian T Unknown Unknown cells and some specialized B cells
CD6 b 3 Mammalian T Unknown Unknown cells and some specialized B cells
Complement Factor I 1 Mammalian plasma Proteolytic regulation Unknown of the complement cascade
Binding the sperm Possibly activating peptide peptide speract binding
Speract receptor 4 Sea urchin sperm
aSee Ref. 6 for detailed citations. bSee Ref. 30.
142
Some of these molecules are not natu- ral, physiologically relevant ligands, but simply share with natural ligands struc- tural features required for high-affinity binding. While these features are present in many different molecules, receptor- ligand binding is not completely pro- miscuous: there are many molecules which cannot bind directly to the receptor and compete effectively for the binding of known ligands 2,7.~. For ex- ample, molecules that do not bind include native and methylated LI)L, native bovine serum albumin, poly(C), heparin, phosphatidylcholine, and many others. The only common feature of scavenger receptor ligands recognized to date is that they comprise poly- anionic monomers or macromolecular complexes; however, there are many polyanions which are not iigands. The broad binding specificity of the scav- enger receptor and its expression by macrophages 2 suggest that it may play a role not only in atherogenesis, but also in other physiological and patho- physiological systems, e.g. macrophage- associated immune responses and inflammation ~,6.
To facilitate further analysis of the functions of scavenger receptors, we and others set out to purify and charac- terize the scavenger receptor molecule. Dressel, Via and their colleagues l°,u and Wong et al. ~2 first reported the partial purification of 200-260 kDa ligand bind- ing proteins from cultured cells and tis- sues. We used a two-step ligand-affinity and immunoaffinity chromatographic procedure to purify scavenger recep- tors approximately 240 000-fold from bovine lung membranes 13. The purified bovine scavenger receptors comprise a set of related N-glycosylated proteins: ~220 kDa trimers, ~150 kDa dimers and ~77 kDa mohomers. The trimers and dimers are converted into the mono- mers on reduction. We could detect ligand binding to the trimeric form, but not the dimeric or monomeric forms.
Based on partial amino acid se- quencing, two scavenger receptor cDNAs, designated type I and type lI, were cloned from a bovine lung cDNA library TM. These were derived from al- ternatively spliced mRNA products a of single gene. Transfection of COS 5,~4 or Chinese hamster ovary (CHO) cells 8,~s with either of the cDNAs confers on them receptor activity exhibiting the distinctive, characteristic broad ligand specificity of macrophage scavenger receptors. Type I and/or type 1I scav- enger receptor expression has been
TIBS 17 - APRIL 1992
detected in macrophages in v i tro s and in vivo 16,17 and in stimulated cultured smooth muscle cells 18 (M. Freeman, pers. commun.). The deduced primary structure of the bovine type I scavenger receptor 5 suggests that the receptor is a 453-amino acid integral membrane protein comprising six discrete domains, while the sequence of the type II recep- tor, which will be described in detail below, is a truncated form with 349 amino acids. The domains of the type I receptor include: I, the amino-terminal cytoplasmic domain (amino acid resi- dues 1-50); II, a single transmembrane domain (51-76); llI, a short spacer do- main (77-108), and three additional extra- cellular domains, domains IV (109-271), V (272-343) and VI (344--453). Domains Ill and IV contain two and five potential asparagine-linked (N-linked) glycosyi- ation sites, respectively.
Extracellular coiled-coil domains The most unusual feature of the scav-
enger receptor's predicted structure is the presence of two distinct coiled-coil extracellular domains. The first, domain IV, contains as many as 23, seven-amino acid or 'heptad' repeats in which resi- dues in the first (a) and fourth (d) pos- itions frequently have aliphatic side chains (leucine, isoleucine, valine). In other proteins similar heptad repeat- containing domains have been shown to fold into right-handed amphipathic (z-helices with 3.5 residues per turn '9. These helices assemble into two- or three-stranded parallel bundles in which the helices wrap around each other to form a left-handed supercoiled fiber held together, in part, by an inter- helical hydrophobic core of aliphatic residues at the a and d positions. Figure 2 illustrates the folding of a single hep- tad into two turns of (~-helix and the relationships of the residues in two- stranded ('leucine zipper') and three- stranded (z-helical coiled coils. It also lists ~xamples of proteins containing these structures. Based on our obser- vations that scavenger receptors sub- units associate as functional trimers ~3, we have proposed 5 that the (~-helical coiled-coil domain may fold into a three-stranded fiber which could be as much as 230 ]~ in length. Imperfections in the heptads 5 may result in the super- helix having discontinuities and its being somewhat shorter. More complex, al- ternative models involving two stranded (z-helical coiled-coils are also possible 5.
The second coiled-coil domain, domain V, contains 24 uninterrupted
Gly-X-Y triplet repeats in which 14 of the Y residues are prolines or lysines. This sequence suggests that domain V forms a classic right-handed collagen- ous triple helix approximately 200 A long. Secreted molecules containing collagenous triple helices include the extracellular matrix collagens 2° which contain very long stretches of Gly-X-Y repeats (e.g., approximately 300 triplets for type I collagen) and the smaller mammalian proteins with 19-61 triplets per chain, including complement factor Clq, pulmonary surfactant apoprotein, mannose-binding protein, and conglu- tinin (see Refs 5 and 21 for detailed ci- tations). The asymmetric form of acetyl- cholinesterase 22 and the bacterial enzyme pullulanase 23 also contain col- lagenous domains. A distinctive feature of mammalian collagens is that the residues in the Y position are often pro- line or lysine and that some of their side chains are hydroxylated post- translationally in the endoplasmic reticu- lum (ER) prior to triple helix formation (see Ref. 24 for additional citations). Proline hydroxylation stabilizes the triple helical structure. We have recently observed the hydroxylation and trimer- ization of bovine scavenger receptors expressed in CHO cells 24. Efficient trans- port of these receptors from the ER to the Golgi required hydroxylation. These data provide direct support for the pre- diction that domain V folds into a col- lagenous triple helix and suggest that stable triple helix formation is required for efficient ER to Golgi transport. The scavenger receptor is apparently the only integral membrane protein con- taining a collagenous domain to be described to date.
A new cysteine-rich repeat In the type I bovine scavenger recep-
tor, the carboxy-terminal extracellular domain, domain VI, contains six of the eight cysteines and is designated the scavenger receptor cysteine-rich, or SRCR, domain. Such cysteine-rich do- mains are found in many cell-surface receptors. Comparisons of the se- quences of these domains, especially the spacings between cysteine residues, has permitted their classification into different families 6,2s,26. Examples include the EGF-like domain, immunoglobulin superfamily domains, the LDL recep- tor/complement C9 domain, clotting factor Kringle domains, and type I and type II fibronectin domains. These di- sulfide crosslinked domains apparently fold into compact structures which
(1) can withstand the rigors of the extra- cellular environment, (2) are well suited for a variety of biochemical tasks, and (3) are readily juxtaposed to other pro- tein domains to allow the constructioa of complex mosaic proteins. It seems likely that exon shuffling is responsible for the generation of such mosaic pro- teins. In many cases the specific func- tions of these domains have not been defined; however, the seven extra- cellular, amino-terminal cysteine-rich domains of the LDL receptor have been shown to compose that receptor's ligand- binding site 3.
Sequence analysis of the bovine and murine SRCR domains revealed that this domain helps define a previously unrecognized, ancient, and highly con- served family of cysteine-rich protein domains 6 (see Table I). The similarities in sequences between the SRCR do- mains of the mammalian scavenger re- ceptors and a receptor from sea urchin sperm, the speract receptor, are es- pecially striking. The scavenger recep- tors' SRCR domains are as similar to each of the four speract receptor's SRCR domains as the speract receptor's SRCR domains are to themselves, with the greatest similarity being 48% se- quence identity with an alignment score of approximately 15. About half of the ~100 positions in all of the SRCR domains examined to date have either identical or highly conserved residues. The functional relevance, if any, of the appearance of SRCR domains in these diverse proteins is unknown. However, it seems likely that the conserved cys- teines within each SRCR domain form three intramolecular disulfide bonds and that the domains fold into similar, probably globular, structures.
Trimeric model of the scavenger receptor's structure
Based on our analysis of the type l bovine scavenger receptor's primary sequence 5 and the results of studies with the purified receptor 13 and meta- bolically labeled receptors expressed in CHO cells 24, we propose the models in Figs 3 and 4 for the structure and biosynthesis of the type I scavenger receptor. In the model of the type 1 scavenger receptor in Fig. 3, the extra- cellular domains of the homotrimeric integral membrane proteinoare illustrated as an approximately 430 A long, coiled- coil, fibrous stalk which is perpen- dicular to the plasma membrane and upon which sit three carboxy-terminal globular SRCR domains. The stalk com-
143
TIBS 17 - APRIL "1992
Rgure 3 Model of the type I macrophage scavenger receptor. This trimeric model for the bovine type I scavenger receptor illustrates the six distinctive domains of the receptor with the number of amino acids in each domain indicated in parenthesis. They are the: I. amino-terminal cytoplasmic domain; II. transmem- brane (TM) domain; III. spacer domain; IV. c(-helical coiled-coil domain comprising heptad repeats with aliphatic amino acids (A) in the first and fourth pos- itions; V. collagen-like domain comprising 24 Gly-X-Y repeats; and VI. scavenger receptor cysteine-rich domain (SRCR) containing six of the eight cysteines in each chain. In this illustration, coiled-coil domains IV and V are overwound to emphasize their triple helical structures. Cys83 in the spacer domain participates in disulfide bonds which covalently crosslink two of the three chains into dimers. Cys17 in the cyto- plasmic domain does not participate in disulfide bond formation in intact cells, but its side chain oxidizes and forms artifactual disulfide crosslinks when cells are disrupted in the absence of a sulfhydryl trap. The trimers might associate to form higher order oligo- mers (see text). Alternative models are discussed in Ref. 5.
prises 52% of the entire receptor and 62% of its extracellular domains. The colinearity of the mhelical coiled.coil domain (left-handed supercoil) and the collagenous domain (right-handed super- coil) and their orientation relative to the plasma membrane are hypothetical. Trimers similar to those in Fig. 3 might aggregate into higher order oligomers, such as those observed for mammalian proteins with similarly short collagen- ous domains, including Clq, lung sur- factant apoprotein and the mannose- binding protein (for review see Ref. 21). These proteins form distinctive struc- tures which resemble bouquets of
144
tulips having bent collag- enous 'stems' and carboxy- terminal globular 'bulbs'.
A model for the biosyn- thesis of scavenger re- ceptors is shown in Fig. 4. Newly synthesized recep- tors subunits are inserted into the membrane of the ER and are subjected to N-linked glycosylation and hydroxylation of prolines and/or lysines in the lumen of the ER. The pre- cursor monomer chains rapidly oligomerize into trimers, each comprising a monomer and a Cys83 (spacer domain, see Fig. 3) disulfide-linked dimer. The precursor trimers are transported to and through the Golgi appar- atus and trans-Golgi net- work where their high mannose N-linked oligo- saccharides are converted into complex, endoglyco- sidase H-resistant sugars. The mature receptor trimers are then trans- ported to the cell surface, where they can partici- pate in the receptor- mediated endocytosis of ligands such as AcLDL, presumably via coated pits and vesicles. Covalent bond formation between Cys83 residues is not required for the assembly of active receptor trimers. In Cys83 -~ Gly83 mutants, the monomers form non- covalently associated trimers in the ER. They are transported to the cell surface where they
bind ligands and mediate endocytosis in a manner indistinguishable from that of wild-type receptors 24. The apparent masses of the precursor and mature forms of the bovine type I and type II scavenger receptors are listed in Table II. If an appropriate sulfhydryl trap, such as iodoacetamide, is not present when cells are disrupted, Cysl7 residues in the cytoplasmic domains (Fig. 3) are oxidized and efficiently form intermol- ecular disulfide bonds. These artifactual disulfides link the non-covalently as- sociated monomers and dimers into covalently crosslinked trimers and are apparently responsible for the reduc-
The model in Fig. 3 raised the possi- bility that the SRCR domains at the end of the fibrous stalk might be involved in ligand binding, especially in the light of the known ligand-binding function of the cysteine-repeats of the LDL recep- tor 3. Although in vitro mutagenesis pro- cedures appeared to be obvious ap- proaches to address the question of the role of the SRCR domain in ligand bind- ing, they were unnecessary. In the course of cloning the cDNA for the type l bovine scavenger receptor, a second, related cDNA was isolated and charac- terized 14. This type II scavenger recep- tor cDNA encodes a protein which dif- fers from the type I receptor only in that the 110-amino acid SRCR domain is replaced with a six-amino acid carboxyl terminus. In CHO cells, the synthesis, oligomerization, post-translational pro- cessing and transport of the type lI bovine scavenger receptor are virtually identical to those of the type 1 recep- tor 24. Both type I and type II scavenger receptors are expressed in macro- phages and both corresponding cDNAs have been cloned from cattle TM, mice 6, humans ~6 and rabbits (M. Freeman, pers. commun.). The two different receptor types are generated by alternative splic- ing of a message encoded by a single gene mapped to routine 6 and human ~6 chromosomes 8. Despite the truncation, the type II receptor homotrimers medi- ate endocytosis of chemically modified LDL with high affinity and characteristic broad specificity, similar to that of the type I receptor ~J4JS. All known scav- enger receptor ligands tested to date bind to both types of receptor.
Ligand binding Analysis of the type II scavenger
receptors established that the SRCR domain does not play a significant role in establishing the distinctive broad binding specificity of scavenger recep- tors. Thus, one or both of the fibrous domains must be directly involved in ligand binding. We previously suggested that the collagenous domain probably plays a key role in ligand binding 5. This was based on the broad binding proper- ties of other collagen containing mol- ecules and the observations that at physiological pH all of the GIy-X-Y trip- lets in the bovine scavenger receptor are neutral or positively charged while its ligands are polyanionic. Recent mutagenesis experiments (S. Acton, M. Freeman, Y. Ekkel and M. Krieger,
TIBS 1,7 - APRIL 1992
unpublished; Ref. 27) pro- vide strong evidence that the collagenous domain is nec- essary for ligand binding. Additional experiments from our laboratory suggest that the collagenous domain may be sufficient to generate the broad binding specificity characteristic of scavenger receptors (D. Resnick and M. Kxieger, unpublished). There is an attractive mech- anism which may explain both the receptor's high affinity for ligands and its broad ligand specificity. The collagen triple helix may provide a selectively sticky surface which ex- hibits broad specificity at low affinity for monovalent interactions, while poly- valent ligand binding either to multiple sites on a single collagen triple helix or to multiple collagenous do- mains in higher-order recep- tor oligomers (see above) may result in high-affinity binding. The scavenger re- ceptor may be acting as a kind of molecular flypaper.
Scavenger receptor lig- and binding is unusual not only because of its broad specificity, but also because it exhibits non- reciprocal cross-competition (i.e. one ligand efficiently competes for the bind- ing of a second ligand while the second ligand only partially competes for tile binding of the first). Non-reciprocal cross-competition of AcLDL and OxLDL was first observed in macrophages 28,29 and subsequently in CHO cells ex- pressing either the type I or type II bovine scavenger receptors ~s. Possible explanations for the non-reciprocal cross-t:ompetition include the presence of multiple, interacting binding sites on a single receptor or the existence of multiple receptor conformations with differing binding properties (see Ref. 15 for further discussion). Elucidation of the mechanism underlying this curious phenomenon will require additional investigation.
What are the normal physiological functions of the scavenger receptor?.
The physiological and pathophysio- logical functions of the scavenger re- ceptor have not been established with
Rgure 4 Scavenger receptor pathway. Scavenger receptors are synthesized 24 as integral membrane proteins in the endoplasmic reticulum (ER), where they are covalently modified by asparagine-linked (N-linked) glycosylation in the spacer and (z-helical coiled-coil domains and by hydroxylation of prolines and/or lysines in the collagenous domain. In the ER, disulfide crosslinked dimers at Cys83 (bovine scavenger receptor) are formed, and dimers and monomers non-covalently associate to form trimers. Trimers are transported to the Golgi apparatus and trans-Golgi network (TGN) for additional processing and sorting to the cell surface. Receptor-mediated endocytosis of ligands such as chemically modified LDL is pro- posed to occur via the coated pit-coated vesicle pathway previously described for the LDL receptor 3. In brief, after high-affinity binding of ligands to the receptor, invagination of cell-surface coated pits containing the receptor-ligand complex results in the formation of coated endocytic vesicles. These are converted to endosomes. The low pH in the endosomal lumen induces receptor-ligand dissociation, with consequent recycling of the receptor to the cell surface and lysosomal digestion of the ligand. If the ligand is modified LDL, this endocytosis leads to massive release of cholesterol and its subse- quent conversion to cholesteryl ester droplets in the cytoplasm (foam cell formation).
certainty. It seems likely, although it has not yet been proven, that macro- phage scavenger receptors are involved in foam cell formation during athero- genesis 2,4. We have found that scav- enger receptor expression in ClIO cells can lead to their conversion to lipid- laden foam-like cells when they are incubated with modified LDL in cul- ture Is. Also, scavenger receptor mRNA and protein have been detected in atherosclerotic plaques 16,]7. Because of their broad binding specificity, scav- enger receptors may also be involved in other physiological sys- tems, especially those involving macrophage- associated host defense activities, such as patho- gen clearance and partici- pation in immune reac- tionsS,E In this regard, we have helped establish Type l wi th Hampton, Raetz and colleagues 8,9 that bacterial Type II endotoxin (lipopolysaccha-
ride), which causes endotoxic shock, is a ligand of the scavenger receptors. Although endotoxin binding to scav- enger receptors apparently does not play a direct role in the signaling path- way responsible for activation of macrophages, scavenger receptor2medi- ated hepatic uptake of endotoxin in vivo plays a quantitatively significant role in endotoxin clearance 8. Thus, scavenger receptors presumably play an important role in protecting the body from endotoxic shock during Gram-negative bacterial sepsis. The
TaMe II. Apparent molecular mass of the precursor and mature forms of types I and II bovine scavenger
presence of the fibrous and SRCR domains in the receptor raise the possi- bility that it may also be involved in cell-cell or cell--extracellular matrix interactions.
The isolation and characterization of the type I and type II macrophage scav- enger receptor's cDNAs have begun to provide tools which should be useful for future studies of the structure and properties of this receptor and its role in macrophage functions in general, and foam cell formation and atherogenesis in particular.
Acknowledgements 1 thank my many colleagues and col-
laborators for their contributions to the studies reviewed here and R. Rosenberg and P. Schimmel for helpful discussions. I also thank M. Freeman for permission to cite results of his labora- tory's unpublished work. This work is supported by grants from the National Institutes of Health Heart, Lung and Blood Institute (]-IL41484) and Arris Pharmaceutical Corporation.
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3 Goldstein, J. L. and Brown, M. S. (1989) in The Metabolic Basis of Inherited Disease, (6th edn) (Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., eds), pp. 1215-1250, McGraw-Hill
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