CHEMISTRY OF HIGH DENSITY LIPOPROTEINS D. N. RAMAKRISHNA RAO AND S. MAHADWAN

(Dept. of Biochemislvy, Ilidialt ZnSlitlIte of Scieme, Bungalore 560012)

Received on June 20, 1977; Revised on August 11, 1977

Hunran serum lipoproteins have been classij?ed into jive major cIasres tramely, cltylomicuons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), nigh density lipoproteins (HDL) and very high density lipcpoteins (VHDL). WhVe ;hestructure and fun ; tbn o f thcse lipoprotems have been reviewed elsewhere, nu ejort has been mane tc review rhe progreqs made in the understanding of the structure of serum HDL obtarned fi.om variuiortr mammalian and non-mammaliai~ pecies. Iiz this review un attcmpt has been made to describe the physiml and cfiemical characterirtrcs of serian and egg yolk high density lipoproteins.

Key Words: High density lipoproteins, Lipovitellins

Lipoproteins can be classified into two major types, the structural lipo- proteins and the soluble lipoproteins. Examples of structural lipoproteins are'lipoproteins of membranes, such as those of plasmillemma, mitochon- dria, chloroplest, myelin and bacterial plasma membrane. The soluble lipoproteins ma.y be classified into three groups namely, milk lipoproteins, serum lipoproteins and egg yolk lipoproteins.

Lipoprotein of cow's milk occur almost excl~~sively as constituents of the fat soluble membrane, a complex lipid-protein system oriented at the fat/plasma interface. Hayashi and Smith [I] have reported the isolation of a high density lipoprotein from cream globule by treatmen.t with deter- gents or by mechanical disruption. Not much structural studies have been carried out on these lipoproteins.

Human serum Zboproteins

Most of the work done in the field of serum lipoproteins concern pre- dominantly with human setum. However, studies of serum lipoproteins

1 22 D. N. RAMAKIWHNA RAO A N D S . MAHADEVAN

on species other than human have also been reported in recent times. A number of reviews have been published concerning the structure and fullclion of serum 1ipoprotein.s 12-41. Human. semm lipoproteins h.ave beer. classi. fied into five classes b ~ s e d on electrophoretic mobility or by their rate of ultracentrifugal sedimentation or flotation in salt solutions 151. They are chylomicrons, very low demity lipoproteins (VLDL) or pre /?-lipoproteins, low den.sity lipoproteins (LDL) or 6-lipoproteins, high density lipoproteins (HDL) or a-lipoproteins and very high density lipoproteins (V.HDL),

Human sevunz high density l#oproteins

HDL are the sinallest of the lipoproteit~. particles with a diameter range of 90-120 A [6]. Hi~man serum HDL has been classified into three sub. classes: HDL,, HDL, and HDL,. I-IDL* was originally isolated between densities 1.050 and 1,063 gm/ml [7]. HDL, is isolated in between densi- ties 1.063 a ~ . d I . I 2 gm/ml end HDL, betweep. densities 1.12 and 1.21 gm/ml. However, HDLl has been shown to be similar to HDL, by electro. phoretic an.d immunocl~emical properties [7]. , Wide HDL, and HDL, have been fractiomted into several subspecies by differential a.ud ratelzonal ultracentiifugation and by analytical and preparative gel electrofocusing [8-131 not much studies have been carried out on these subspecies. In normal male subjects the concentration of HDL, is about 70 mg/100 ml serum while that of HDL, is 230 mg/100 ml serum. In females concentration of HDL, is about 219 mg/100 ml serum and HDL, is 238 mg/100 ml[14-161. The reason for the sex-d.epen.dent differential distribution ir not clearly know. However, a relation between HDL, and estrogen levels could exist as this class of HDL is thrce times more in premenopausal women than in men [14-161.

The physical charactelistics and protein components of HDLa and HDLa [17]are given in Table I while the chemical characteristics of human HDL are compared with HDL of other animal systems in Table 11. Human serum HDL contains approximately 50% protein and 50% lipid. Phos- pholipids, cholesterylester and triglycerides are the main lipid components. Phosphatidyl choline constitutes 7040% of the phospholipids and sphingo- myelin 12-14%. Phosphatidyl serine arid phosphatidyl inositol are ~ o T

corqponents. Linoleic acid is the predominant fatty acid of the cholester~l ester [2].

Very high density lipoproteins

VHDL is isolated between densities 1 .Z1 and 1 .25 gm/ml. It is present at a concentration of 20 mg per 100 ml serum. The physical characterisks

C%enzistry of High Density Lipopmieins 123

are compared d t h those of HDL, and HDL, in Table I. VHDL is approximately 60% protein and 40% lipid. Phospholipids are the major components of lipids and cholesteryl ester is a minor component [14].

Apopvoteitz structure of human serum HDL

The protein components of HDL are soluble in water and hence they have been very well characterised. About 90% of the protein moiety of HDL is composed of two major apoproteins which are designated as apoA-1

Physical churacteristiw and proteiiz coi~zponefits of humw serum HDL, HDL,, HDL, and VHDL

Lipoproteins HDL HDL, HDL, VHDL

Density (grnlml) 1.063-1.21 1.063-1.12 1-12-1.21 1.21-1.25

Flotation rare F 1.2-0.20 F 1.2 3.5- F 1.20-3.5 . . 9 . 0

Molecular 170,000 360,000 170,000 154,000 Weight 360,000

Apoproteins ApoA-I ApoA-I ApoA-I (70%) (6%) (74D

ApoA-I1 ApoA- It ApoA-11 (18%) ' (20%) (160j,) .

Th~n Lne protein and

'. As determined by Elcctron mmoswpy. Data obtained from references [2, 14, 171.

Lip

id co

mpo

sitio

n" (p

erce

ntag

e by

wei

ght)

R

efer

e~ic

~s

S1.

Ani

nlal

s N

o. 1

2

Muw

nalr

1.

Hum

an

2.

Chi

mpa

nzee

3.

Rhc

sus

mon

key

4.

Pata

s mon

key

5.

(a)

Bw

ine

Typ

es

Prol

ein

Lip

id -

of

PL

CE

UC

T

G

FA

Mix

el.

HD

L

P la

neou

s ?: - 8

HD

L

50.0

50

.0

48.0

32

.0

8.0

10

.0

2.0

..

4,

11

+

HD

L,

43.0

57

.0

50.9

30

.0

8.7

8-

7 1.

7 ..

4, 1

1 H

DL

, 5

60

44

.0

52.2

27

.2

9.0

7

.0

4.5

. .

? 0 H

DL

, 41

.8

58.2

42

.2

42.8

11

.2

3.8

..

. . 76

$ u

HD

L,

57.7

42

.3

45.3

40

.2

8.0

7.0

. .

. .

. . V1

HD

L

32.0

68

.0

27.0

37

.0

6.0

29.0

..

. .

80

(b)

Bul

l(H

eref

ord)

H

DL

30

,O

70.0

24

.6

47.O

**

. . 27

.0

1.4

..

83

6.

Bis

on

7.

Hor

se

8.

Cam

el

9.

Pig

10.

Dog

11.

Shee

p

12.

Rat

13.

Rab

bit

14.

Gui

nea

pig

(a)

norm

al d

iet

HD

L

HD

L

HD

L

HD

L,

HD

L,

HD

L

HD

L,

HD

L

VH

DL

HD

L

HD

L

HD

L

H-3

, 85

M

G-I

, D

G-2

(6)

chol

este

rol r

ich

diet

K

DL

27

.6

72.4

42

.5

21.0

33

.8

2.7

. . . .

99

126 D. N. RAMAK,RISANA RAO A N D S. MAHADBVA~

n".,"""."

.

24.

Frog

R rh

25.

Salm

on

26.

Paci

fic sa

rdin

e

27.

Dog

fish

28.

Hag

fis

h

Inve

rteb

rate

s (in

sect

s)

HD

L

51.0

HD

L

NR

HD

L

NR

HD

L

NR

HD

L

NR

29.

h~

ric

an

sil

k m

oth

HD

L

52.0

* Lip

id c

onlp

osit

iono

f som

eof t

he a

nim

als d

o no

t add

up to

100

%.

The

y ha

vebe

en p

rese

nted

as

repo

rfed

.

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clud

es u

ncst

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ster

.

**' I

nclu

des

fitt

y ac

ids

and

digl

ycer

ides

.

Abb

revi

atio

ns

PL =

Pho

spho

lipid

s U

L =

Une

ster

ificd

chol

este

rol

CE

= C

hole

ster

~les

ter

.

TG

= T

rigl

ycer

ide

FA

=

Fatt

y ac

ids

DG

= D

igly

ceri

des

MG

=

Mon

ogly

ceri

de

H

=

Hyd

roca

rbon

s N

R =

Not

rep

orte

d G

=

Gly

ceri

des

128 D. N. RAMAIGRI~HNA RAO AND S. MAHADEVAN

This is a major apop;otcin constituti~lg about 68% of apoHDL. It has a mnolecular wcight of 28,300 and is a single polypeptide chain. of 245 amino acid residues. Acpartic ncid is the N-terminal amino acid and glutamine is the C-terminal amino ncid. I t does not contain cysteinc, cystine or ]so- leucine. ApoA-I occurs in two polymorphic forms [37] , apoA-1-1 and apoA-1-2 which have slight differences in amino acid composition and electroplioretic mobility. ApoA-I contains about 60-70% a-helix, 37y0 random coil and 8% aiztiparallel p-structure [38]. It has a physiological function of activating an enzyme known as lecithin: cholesterol acyl transferase (LCAT) [39, 401. ApoA-1 may also have a function in regulating the content of membrane lipids [4I-431 and maintaining proper membrane fluidity [44]. The term opoA-I is generally refened to a serum HDL-apo- protein which has a molecular weight of about 28,000.

(ii) ApoA-TI

This protein constitutes about 20% of RDI, proteins. It contains two identical monomers of 77 amino acid rcsidues which are linked through disulfide bridge at residue 6. The N-terminal amino acid is pyrrolidone carboxylic acid and the C-terminal amino acid i s glutamine. ApoA-11 does not contain carbohydrates, histidine, arginine or trytophan. It contains 35% a-helix, 13% /3-structure and 52% disordered structure [38]. The term apoA-IS is referred to a serum HDLapoprotein which has a molecular weight of about 8,500.

(iii) Apo C-I

While apoC-proteins are the major protein components of E D L , it forms about 12% of HDL apoproteins. sZpoC-I is a single polypeptide chain of 57 amino acid residues. The N and C-terminal zmmo acids ,are threonine and serine respectively. It does not contain histidine, tyros%

Chemistry of Higk Density Lipoproreins 129

cysteine or cystinc. I t activates two lipoprotein enzynles namely, LCAT /47] and lipoprotein lipase purified from human postheparin plasma 148, 491.

(iv) ApoC- 11

ApoC-11 contains about 100 amino acid residues with a molecular weight of 12,500 11 is a potent activator of lipoprotein lipase fiom human and rat post-heparin plasma an.d cow's milk [51-541.

(v) Ayo C-111

ApoC-111 is a major component of VLDL containing 79 amino acid residues. However, it is preser?t as a minor component in HDL. ApoC-111 does not contain isoleucine, cystine or cysteine. The N and C-terminal amino adds are serine and alanine respectively. At Threonine-74a carbo- hydrate mo~ety is attached by o-glycosidic linkage which contains one residue 'each of galactose, galactosamine and either 0, 1 or 2 residues of sialic acid per mole of the polypeptide [50]. This corresponds to three polymorphic forms, namely, apoC-111-0, apoCII1-1 and apoC-111-2. ApoC-111 is an inhibitor of lipoprotein lipase at a concentration of 2% (W/W) [55]. This inhibition is not reversed by apoC-I1 which is an activator of this enzyme. Phospholipid binding with four fragments of apoC-111 corresponding to the sequence 41-79, 48-79, 55-79 and 61-79 showed that sequence 48-79 binds significant q~laniities of phosphatidyl choline [56]. Trypsin diges- tion of apoC-IIr-PC complex suggested that hydrolysis proceeds in a facile manner at the N-terminal half, while the C-terminal half is protected from cleavage [57]. Based on thcse observations, the C-terminal region is found to be the phospholipid binding site.

Models of Aunzan serum lipoproteins

Any model describing the structure of serum lipoproteins should consider the problem concerning the solubility of serum lipoproteins in water. Hence models describing the relative location of lipid and proteins are largely influen- ced by the existing knowledge about the structure of miscells. The lipid-rich lipoproteins like chylomicron.~ and VLDL are known to contain a lipid-core structure made of cholesteryl ester and triglycerides. Cholesterol,protein and phospholipids form the outer surface [58, 591. In the case of LDL, two models have been proposed concerning the relative location of lipid and proteins. Based on small angle X-ray data, a phosphol~pid bilayer model consisting of cholesteryl ester, cholesterol and triglycerides has been suggsted.

130 D. N. RAMAKRISHNA RAO AND S. MAHADEVAN

This bilaycr surrounds a central cox of pl'otcins [@I. A slightly dipafferent model consisting of a trilayer structure has been proposed based on NMR spectroscopic studies [GI 1.

Modeis of human serum HDL

Earlier models of HDL are based on electron microscopic studies [62.] Since the publication of small angle X-ray data of HDL, considerable pro. gress has been made in the understanding of the structure of human semm HDL. Small angle X-ray data revealed the existence of two distinct regions in HDL namely, the electron-deficient central core which has a dip meter of 86 A and an outer electron-rich region which has a radius of 14A [63]. Phospholipids can be accommodated in this region since the phos. pholipid head groups, measure about 11 A wide. Proteins are also located on the outer surface and the evidence for the surface location of proteins and phospholipids comes from a large number of experimental data. Almost all the proteins and phospholipids are accessible for enzymatic hydrolysis [64, 651. More than 90% of the amino groups of lysine are available for succinylation reaction [66]. More than 90% of apoA-I is detected by radioimmunoassay [67]. The central core of HDb consists of cholesteryl ester and triglycerides.

The primary sequence of HDLapoproteins seems to have some speci- ficity which permit them to bind lipids. ApoA proteins bind signifcant amount of neutral lipids only in the presence of phospholipds. They also exhibit a significant increase in helical content upon binding phospholipids [681. Hence the interaction of phospholipids an.d proteins is of prim@y mportance. Assuming helical structure of proteins are important for binding lipids, Segrest et al. [@I] have suggested that these proteins have amphipathic helical regions, that is, one side of the helix is polar and the other side of the helix is non-polar. Such an arrangement facilitates the interaction between non-polar region of the internal core and the external aqueous medium simultaneously. Prediction analysis of apw-I sug@ts that the occurrence of at least nine helical regions of approximately equal ength in the central and C-terminal portion of the chain, separated by helix breaking sections containing proline, glycine or both [go]. Each helical region contains 15-20 amino acid residues. This arrangement also causes the helix to be amphipathic [go]. Helical areas between residues 1 1 4 of

apoA-nP0) and residues 7-14, 18129 and 33-53 of apoC-I can also giye raise to amphipathic areas [41]. The polar face of the amphipathic heh of aPOC-1 contains a highly unusual topographical distribution of charged

Ciwnistrj~ of High Density Lipoproteins 131

amino acid res'idues. Glutainic and aspartic acids a*e localised at the centre of the polar face, while positively charged lysine and argin.ine residues are oriented towards the lateral cdges at the interface between the polar and non-polar faces. This arrangement leads to a suggestion ffiat there may be electrostatic interaclion between the positively charged choline groups of the phospholipid with glutanic or aspartic a.zid side chains and between the phosphates and the lysine or argininc residaes. The importance of lysine in the bin-ding of lipid is demonstrated by the abolition of lipid binding by maleylated apoA-11/72]. ApoA-I binds very little HDL lipids. 'However, in presence of apoA-11, apoA-I binds significant amount of total HDL lipids[73], suggesting Lhat portein-protein interaction is also necessary for binding, lipids. Based on these results two modcls have been proposed for HDL.(Fig. 1). In the model of Jackson et al. [74], the long axis of fatty acyl

Fro. 1. Schematic represents .ion of humam scrnm high density lipoproteins A: HDL model of Jackson e ta l . I741 B: HDL model of Assmann and Brewel- [YOI.

hain is perpe~.dicular to the helical protein which i s surlounding the sur- ace. Assmann eta[ . [70] havc proposed a lipid mosaic model similar to hat of membranes proposed by Singer [75]. According to this model roteins are like "ice-be~gs " flozting in a sea of lipids and phospholipid cyl chains are parallel to a-helical portions of the protein, which is in con- cast to the prcvious model. Both these models, however, permit the inter- ction of carbon atoms 2-4 of the h t ty acyl chain of phospholipid with the .ydrophobic side of the helix and possibly with the hydrophobic region ~f lysinc and arginine residues.

Chimpcu?zec serum FIDL has been fi-ncliowtted into HDL, and HDL, and they differ si&~%,ific:ultly in chemical composition [76]. Chimpanzee ape-HDL differs from the human apo- HDL by a significant decrease in ae realativc content of apoA-TI. Amino acid composition of chimpanzee apoA-I is very similar to human npoA-1 t.xcept for glulamic acid, leucine and aspartic acid (Tahlc 111). Chimpmzce npoA-11 is similal to hllman apoA-II exccpt for histidim and nrgininc (Tablc IV). These differences are attributed to small coninmination by 2poA-1. Onc of the interesting features of chimpanzee npoA-11 is thc presence of cystine. So f a cystine containing apoA-ll h a s not ~ C C I T . isolated from any species exccpt human 'and chimpanzee.

Rhesus monkey HDL

Rhesus monkey HDL, and HDL, arc similar to heir hulnan counter- part in terms of hydrodyilamic, spectroscopic and morphological criteria [77]. The ratio of HDL, to T-IDL, is 2 :1 in rhesus monkey as compared to 1 :3 ratio in human serum. The chemical composition of HDL, and HDL, arc described in Table I[. Rhesus monkey apoA-T is very similar to human apoA-I in amino acid composition except for glula~nic acid and arginine (Table-3). It has 9 residues more or glutamic acid and 4 residues less of arginine wh.en compnrcd to human apoA-I(78). Monkey apoA-I1 shows some djfferei~ccs with human apoA-TI (Table-1V). The forinel has been partially sequencccl a t both N and C-terminal ends and the sequence is similar to liun~an apoA-Ti except Sol the subhtitution of swine for cyftcine at positio~l 6, and glutamic acid h lysine a t position 3(79).

Pafus Monkey serzmi BDL

Patas monkcy apoA-I is thc major component of apoHDL while apoA- 11 is a minor one. ApoA-I is strikingly similar to human apoA-1 in amino acid compositiol~ except for 1ysin.c and serinc (Table 111). Partirl sequence of 20 amino acidresidues from the N-terminal end shows that it is identical to that of human apoA-l with the following substitutio~~s : mo~-key-6-tbe@ nine to human 6-serine, monkey-15-valine to human 15-alanine and monkey- 2-glutamic acid l o human-2-itspartic acid. ApoA-TI appefrs lo be similar to the monomeric form of human and rhesus monkey apoA-11 in amino acid composition. Patas monkcy apoA-I1 docs not contain histidine, cysteine is~leu m e ~ n d t~ yptophan,

TABLE I11 Vuriutions in a p A - I of serzlm HDL

Aoimals

Val-idon in amino acids, when cornpared to human apoh- I

An&o acids 2 _ .- R eferences absent Deficient Exccss*

I. Human

2. Chimpanzee

3. Rhehus mo~llccy

4. Patas monkey

5. Bovino

6. Canine

7. Rat

8. Guinea pig

9. Pig

10. Chicken

11. Salmon

Isolcuc nc Cysiinc isolcucinc Cysteine

lcoleucinc Cysteine

Isolecuine Cystcine

Cysteinc

Cy steine

. . Cysteine Psolinc

Cysteinc

Cystcine

Tryptopha~l

. . . . Glutamic acid Aspartic acid Lcucine

Augininc

Lysine

Mctliioni~ie

Ly sinc Thre onine

. . Leu cinc Methionine

Glycine

Aspartic acid Gly cine Alanine Pl~enylalanine H:stidine

Glutamic acid

Serine

Alaninc Isoleucinc

Glycine Isoleucine Alanine Serjne

Isolcncine

Threonine Isoleucine Alanine Aspartic acid

Isoleucine Alanine

Aspartic a ~ i d Lysirre Leucine Methionhe

Tyrosine Alanine Isoleucine

* Amino acids absent in human apoA-I1 but present in other animals aleconsidered excess.

t34 D. N. RAMAKRISIINA RAo AND S. MAHADBVAN

T A B L ~ I V

Varictrions in apoA-ll of serum fIDL

Human

Chimpu,an~ec

Rl~esus monkey

Rat

Salmon

Anzino acids al)scnt .. . ~ . -

Histidim Argin i~rc Tryptophan

. .

Histidint: Isolcucin~= Tryptophan Cystcfnc

Histidinc lsobucii~.u Tryptophan Cystcine

Hist:dinc Tryptophan Cysteine

Cysteine

. . Histidine Arginine

GLycim Arginine

Valine Arginine Lysine Alanine

Aspartic acid

Threoninc Histidine Serine Ar_pin;ne Olutamic acid Aspartic acid PJ~enylalanine Alanine

Methioni~~e Isoleucin~ Glycine

*Amino acids absent in human apoA-I, but present In are considered excess.

Clteinistry of High Density Lipoproteins 135

Baboon serum HDL

Though the chemical composition of baboon selum HDL has not been reported, apoA-1 and apoA-I1 has been studied [78]. While apoA-I1 does not contain cystine or cysteine, apoA-1 is reported to be similar to human apoA-I, though its amino acid composition is not given [78].

Bovine serum HDL

Bovine serum HDL is the predominant lipoprotein which constitutes 80% of the total lipoprotein by weight [SO] and it resembles human HDL,. Bovine HDL has a significantly large amount of triglycerides (Table 11) [81]. Bovine apoA-I constitutes 88% by weight of the apoprotein 1821. The remaining 12% is constituted by two polypeptides of molecular weight 11,000 and 13,000. They resemble human apoC proteins in moleculzr weight and elution position during sephadex gel filtration. Bovine apoA-I is similar to human apoA-I in amino acid composition except for alan-ine and isoleucine whjch are slightly more and methionine being little less. Cysteine is absent.

Hereford bull serum HDL has a single component as show11 by paper electrophoresis and analytical ultracentrifuge methods [83]. It resembles cow serum HDL in having a large amount of triglycerides (Table 11). Bull serum apoA-I is partially sequenced from the N-terminal region [84] and the sequence is similar to that of human apoA-I. The exceptions are, as compared to human apoA-I, glutamic acid in place of pro1in.e a t position 4 and phenylalanine in place of aspartic acid at position 13. Leucine is absent at position 14 [84].

Bison serum HDL

HDL is the major component and it comprises 64% of the total serum lipids [85]. Only the lipid composition of HDL has been reported. An interesting feature of this composition is the presence of hydrocarbons and a large amount of sterol esters (Table 11).

Horse serum HDL

HDL is the major component of horse serum [86]. Presence of large amounts of free fatty acid appears striking (Table 11) and possibly requires confxmation.

136 D. N. RAMAKKISI-INA RAO AND S. MA~XADBVAN

Carnal senon HDL HDL occurs a t a conceniration of 11 mg/100 ml serum [87]. ~t colltains

about 30% protein and 70% lipid (Table 11). NO report on protein fractio. nation has appcarcd.

Pig serum HDL

Porciix HDL, diffcrs considc~nbly fiom HDL, ill. chemical coaposi. tion [88]. ApoA-I i s t l~c nx~jor conzpollcnt of pig apo-HDL and apoA-11 appears to bc abscnt [W]. Pig apoA-I has 261 amino acid residues as compared to 245 residues in h u n ~ ~ n apoA-I. The amino x i d c~mpositjofi~ of porcinc and h~irncm apoA-l ore simiinr except that pig apoA-I contains isoleucine (Table 111). Like h ~ ~ m a n apoh-1, pig apo-1 also activates LCAT. Thc s t ruct~~rc of porcinc HDL studicd by scveral physical methods like small angle X-ray diffixction, NMR and ESR spectroscopy, circular dichorism and differential scanning calorin~ctric methods show that most of the polar groups or phospholipid and protein arc located at the surface [90, 911.

Dog serum HDL

HDL is the nu~jor lipoprotein of caninc swum lipoproteins. It has a HDL fraction tclmcd HDLl which has a density lower than 1 ,063 gm/ml and i t has bcen shown to be similx to high density lipoprotein by imnuno- chemical reactivity, clcctrophorctic mobility and apoprotein content [92]. HDL, does not resemble any high dclasity fraction of human serum. An interesting fcaturc of the lipid and phospholipid composition of canine HDL is that glycerides are ptcscnt a t very low concentrations (Table 11) and phosphatidyl cholinc is ili: major component of phospholipids. ApoA-I of callinc HDL conslitutcs,g0% of spo-HDL, i t has 243 amino acid resi- dues as comparcd to 245 residues of human apoA-I. They are similar in amino %id composition with the exception of isoleucine, alanine and histidine [93]. Canine apoA-I has becn partially sequenced from the N. terminal end and the sequence is sin~ilar to that of human apoA-I. The only exception being the absence of proline a t position 4 [%I. &nine a P o - m L has a polypeptide of nmlecular weight 8,000 which appears to be analogous to human apoC proteins [93].

Sheep serum HDL

Sheep serum HDLL has been isolated between densities 1.063 and 1.075 gm/ml and HDL, between densities 1.075-1.20 gm/ml and VmLat

Clzernistry of High Density Lipoproteins 137

a density greater than 1.21 gm/m1(95). HDL, constitutes 5% of total serum lipoproteins and HDL is characterised by a large amount of cholesteryl ester (Table 11).

Rat serum HDL

Rat apoA-I is the major component of apo-HDL and constitutes,about 62% by weight of HDL(96). The emino acid composition of rat apoA-I is similar to human apoA-I (Table IIJ) except isoleucine (22). Rat apoA-I1 is similar to human apoA-11, with both having pyrrolidone carboxylic acid a t amino terminus, both lacking histidine and tryptophan and both being particularly rich in glutamine andlor glutamic acid. Rat apoC-I is insoluble in acidic buffers. I t laoks tyrosine and cystein.e but contains histidine and is rich in lysine. I t does not contain sialic acid, or hexosamine. Rat apoC-I1 contains histidine but does not contain sialic acid or hexosamine. It is similar to human apoC-I1 in amino acid composition. Like human apoC-11, rat apoC-I1 activates lipoprotein lipase [97]. Rat apoC-111 is found in a t least two polymorphic forms that differ only in their carbo- hydrate content. One of the two polymorphic forms of rat apoC-111 is designated as rat apoC-I114 which does not contain sialic acid or hexo- samine, while the other major polymorphic form, apoC-111-3, contains one residue of galactosamine and three residues of sialic mid. No trisialylated apoC-I11 has been described among human apolipoproteins. An apoC- 111-0 which does not contain sielic acid has been identified in human serum VLDL. I t is, however, not determined whether it also lacks hexosamine. It is also significant that both rat apoC-111 and human apoC-111 could not be distinguished from rat apoC-11 on alkaline polyacrylamide gel electro- phoresis. Other significant compositional differences between rat and huumsn apoC-I11 include high glycine content, presence of isoleucine and absence of histidine in the former. Rat apoC-I11 is unique' in containing C-terminal proline [97].

Rabbit serum HDL

It occurs a t a concentration of 160 mg/100 ml serum and cholesteryl :ster is the major component of lipids 1871.

. . . Guinea Pig serum HDL ,8t

HDL is found in small amounts with LDL being the major compo- lent. The distribution of these lipoproteh is highly dependent on the

Content of the animal diet. When this is low, the principal lipoprotein

138 D. N. RAMARRISHNA RAO AND S. MAIIADEVAN

has w hydrated demity close to 1 ,063 .fP/nll. But at higher levels tb-e h,ydp.ted demity falls less th2.n 1.03 gmlnll. The decrease in decsity is eccomparr.ied. by en increase in molecdx weight, paticle diemeter wd in the ratio of lipid to protein [98]. In animels fed with no1.mz1 diet, the con- centration of HDL is low while with cholesterol rich diet, the levels of HDL is hi&. Chemical ~ompOSiti01. of HDL obtaimd from animals fed on normal diet was 39% protein and 61% lipid. TI-. ~h~leskrol-rich diet aninpls, th.e protein constituted 27% md lipid. 72% [99]. Th-e lipid composition of

the two HDLs me given in Tzble 11. Guima pig 7.poHD.L h2.s beer! fr2.c-

tioneted into 6 fractions [100]. One of these fiectiol?~ resembles h . ~ m ~ . ~ , apoA-T but differs in not having prolil?e but cont,?i~ir?g isolcucir?e. ~t cont2.in.s less of leucine ar.d large anount of almi~2.e. Guir.ezpig 2,~oc-r differs from h.unti?n 3.poC-T in not having proline but it co1dair.s slllaller amounts of histidine m d iyrosir.e, 2.n.d larger amounts of glycine apd alanine [98].

Hegde hog serum HDL

HDL is present at e concen tmtion of about 430 mg/l CO nd selLm m d it has 41% protein and 59% 11pd [87].

Dolphin scrum HDL

HDL is chc miljor lipoprotein pyesent in 1b.e swum of pimipeds (seals) 2nd. bottle-tlose dolphins. AP. inieresljrg feeturc of HDL is th?.t phos- pholipid is the mp.jor compor.ent of lipids in pimipcds whereza cholesteryl ester is the major lipid in do1phir.s [99].

Whale serum ZIL)L

Killer whele HDL constitutes 50-70% of tl.e scrbm lipoproteins erd cholesteryl estcr is thc major comporent of lip~ds [I001

Aria)? serum HDL

Chicken serum HDL

Avian HDL is isolated between the szme densities (1.063-1.21 &m!ml) as humm serum HDL. Rooster serum has sign$car.tly more HDL than chicke~ serum. Immunochemically three distir.ct lipoproteins h m been distinguish.ed. They are VLDL ertd LDL, HDL and lipovitellir.s [loll. HDL is the o~.ly serum 1ipop:otein componeilt which shows similW' to the human serum HDL. Chemical composition of rooster HDL resembles

Chemistry of High Density Lipoproteins 139

that of human HDLa and physical properties of rooster HDL such as partial specific volume, frictioml ratio, hydrated or unhydrated density m d mole- cular weight resemble that of human H D b .

Chicken apo-HDL consists of about 85% of apoA-I and the rest of them are zpoC protejm [1021. Apo-11 is absent. Chicken epoA-I coo- t h s 234 arrin.0 acid residues and. it is similar to h u m qoA-I in. amino aad composition. with the exception of isoleucine, histidine, leucine and glycine. M-terminal region of chicken apoA-I has been sequenced 182, 1021 and it differs considerably from human apoA-I. A striking feature of chicken. apoA-I is tb.zt the protein con tab a large amount helical con.- tent (about 90%) [102]. The helical content of human apoA-1 is about 65%.

Ofher avian serum HDL

HDL of duck, guinea-hen, pheasant md qail serum have been used for immunochemical studies 1103, 1041. There is no report on the physical or chemical characteristics of these lipoproteins or their protein components.

Chemical composition of HDL of pigeon and goose'.have been reported. The interesting feature of HDL in these two birds js that it is present in the serum at a high concentration. Goose serum has 761 mg HDLJ100 ml serum and pigeon has 1352 mg/100 ml serum [87].

Reptilean and amphibian serum HDL

HDL of four species of reptiles an.d one species of amphibian have been isolated and their chemical compositions are given in Table I1 [87]. Fractionation of their protein components have not been reported.

FtSh serum HDL

Salmon serum HDI;

HDL is the only lipoprotein presen.t in the serum of late pre-spawning pink salmon. In contrast to this, the early pre-spawning salmon, con-s both LDL and HDL [105]. Salmon HDL is similar to human HDL-in physical properties like flotation rate, hydrated density and CD spectra. Sallnon apoHDL contains both apoA-I and apaA-I1 protehs. Both of them differ considerably from their human counterpart. Salmon apoA-I does not contain tryptophan but it has much less of aspartic acid and ieucbe, considerably more of 1ysj.q methionhe and tyrosine and much more of

&nine, and isoleucine than human apoA-I. Salmon apoA-11 does not 'contain cystine but contains much less of lysine, th.reonine, serine, acid ar.d phen.ylalanine and considers?bly morc of histine, argbin.e, aspartic acid, glycine, a1an.ine and isoleucine than human apoA-I1 [105].

Sardine serum HDL

Three species of HDL have been isolated from pacific sardine serum namely, HDLL (between densities 1.04-1 .09 gmlmi), HDL, (between densities 1.09-1 .I2 gmjml) and HDL, (between densities 1.12-1.21 mlmi) [106]. HDL, is more abundant than HDLz. NDL ha also been isolated from the semn of.dog fish and hag fish [871. An interestmg feature of rhe 1,pid composition of sardine serum HDL (Table 11) is the absence of'long chain fatty acid, which was expected to be present as these a~irnals feed mainly on plankton rich in wax esters.

. ,

invertebrates

Insect hemolymph HDL . .

HDL is the major lipoprotein of american silk moth Hyalophora cecropia- - It is isolated between densities I .156-1.170 gm/ml. and VHDL has been isoleted et 1.26 gm/ml density [lo?]. Hemolymph HDL has also been isolated from other insects [108, 1091. In American. silk moth, HDL occurs at a concentration o f . 4700 560 .mg/l00 ml, It contains 52% protein and 48% lypid and VHDL contains 94% protein. Phosph.atidy1 ethanolamine and digfycerides ere tne major components of lipids. Diglycerides consti- tute 69% of the total neutrel lipids. It is suggested that hemolymph HDL may. be the carrier of diglyoerides in insects. Disc gei electrophoresis of HDL shows the presence of two major protein components.

- . . -. . . . General remarks about serum lipoproteins

Some mammals like man possess a t least two classes- of lipoproteinh the3 low density 'and high density types, while others have only one type. .Mammals like guinea.pig, earnel. and.rabbit have.very little HDL [ST, 971, while cow, :bison, horse, dog, dolphin and whale have HDL. as the main ,lipoprotein [80, 84, 85, 92, 99, 1001. . Moreover the pattern of .serum lipo- proteins within a species m y be influen-ced by strain or variety. This diffe- rence is seen in salmon, .where in early pre-spawning pink salmon both HDL and LDL are present, .whereas in late .pre-spawn.ing type only HDL is present. tBy irWnundchernica1. reactions i t has been,.shown that there is, no cross

Chemistry of FIigIz Density Lipoproteirrs 141

reaction. between the HDL of animal species belonging to different classes [103, 1041.

Chemically, mammaha11 !czuln HDL does not differ much from that of man. Thc gencral feature of mammalian serum HDL is that it conta i~ .~ 50-70% lipids and 3 0 ~ 50% proteins. Ch.olestero1 is present mostly in. th.e osterificd form and triglycerides fonn less than 10% of th.e lipids. phosph- tidy1 choline is the major phospholipid. The major HDL-apoprotejn namely, apoA-I, has a molcculur. wejgl!t of a.bout 28,000. 11 has a helical content gieater th.an 50% in the intact lipopr.oteil?. It usnally lacks cy,teine. Glulemic acid comprises nearly 20% of the total m1i17.0 acids. An interest- ing fcztuie of qoA- I is that isoleuci11.e is absent in human an.d primates, but is present in. all other spccics. Isoleucjne is present in. human zp.poA-11 but it is abscnt in rhesus and palas monkey. Mammalian apoA-I does l i o ~ contaii~. carbohydrates. ApoA-I1 lacks histidine and tryptophan with the exception of the chiinp'~uzee. Cystine or cystei~~e is also absent in mammalian apoA-I1 with the exceptions of human and chimpanzee (Table IV).

HDL from non mammalian spccies also contain. 50-60% lipid and most of the cholcste~ol exists in the form OF esters. However, in a primitive form of fish, the bag fish, most of the cholesterol is foul~d ill the unesterified form. In a!,. irweriebrate, such as American silk moth unesterified cholesterol js somewhat more than esterified cholesterol. The striking feature in insect NDL is the presence of large amo~rnts of diglqcerides. Unlike few higher animals this insect has lipoprotein of density greater than 1.21 gm/ml.

Egg yolk lipoproteins have been isolated from certain oviparious verte- brates [I 10-1 161 and a few invertebrates such as crustaceans [I 17-121 1. Avia~?. egg yolk contains two types of lipoproteins, the very low density lipop~oteins (VLDL) or lipovitcllin?n and the high density lipoproteins or lipovitellins (Lv). The high and low den.sity cl~.sses in the a ~ i a n egg yolk can bc separated on a solvent density between 1.00 and 1.2 gm/ml [I 1. Egg yolk of frog and fishes do not contain well defined VLDL [122, 1231. Egg yolk of invertebrates do not contain YLDL.

Avian Egg ,Yolk . . ,

Hen's egg yolk ggranvles . . Egg yolk granules consti t~~te 23% of yolk solids and they are co3nposed

3f lipovitellins (Lv), pllosvitin and a small amount of low density lipoprot&n

i4z D. N. RAMAKRISHNA RAO AND S. MAHADEVAN

(LDFG), which collstitute about 4% of the total yolk lipids [124]. Lipom "itellins consist of two components llanlely, a-an.d P-lipovitellins and each has a dimeric molecular weight of 380,000'-400,000. Alth.ough ffractionation of these two have been achieved by TEAE-cellul~se column ~ h ~ o m a t ~ ~ ~ ~ ~ h ~ [125-126], they are similar in man.y respecis. Th.ey have similar a m o acid a.~.d lipid composition [127]. a-Lip~~itellill has 17% and P-lipovitel]in hss I 8.5% lipid [1261. The lipid composition of a a-lipovitel~in is, 60% phospholipids, 36% triglycerides and 4% cholesterol [124], whereas p-lipovitelliil has 62% phospholipids, 34% triglycerides and 4% cholesterol, Cholesterol is present mostly in unesterified form. Phospholipid compo. &ition of a- and P-1ipovitellin.s are given below [124].

Percentage of phospholipids

a-IJipovitelIin P-Lipovitellin

Phosphatidyl choline 75 76

Phosphatidyl ethailolamine 18 17

Ly soleci thin Lysocephalin Sphingomyelin 3

Although lipovitellins have been characterised in a few species of birds, ~eleosts and crustaceans, their protein components are poorely understood. Franzen and Lee [I331 reported the presence of two protein subunits of hen's egg yolk a- or 8-lipovitellins, but detailed i~.vestigations were not carried out to characterise these two proteins. 117. view of limited information on these protein subunits, a detailed study of the protein components of hen's egg

' 1ipovitellin.s were un.dertaken in our laboratory [I321 an.d a brief desaip- tion is given here. The protein components of each of the two hen's egg lipovitellins were fractionated into two protein fractions. a-Lipovitellin was fractionated into a, and a, fractioiis and 8-lipovitellin was fractionated into PI a ~ d 13,, fractions. a, and p, fractions were homogeneous .each -having a molecular weight of about 30,000. a, and PI fractions were hetero- geneous containing more than one protein. A11 the four protein fractions contained neutral sugars, hexosamines, N-terminal lysine and phosphoNs. Sialic mid was present only in a, protein fraction. a, and ,R, firactions were rich in neutral sugars and phosphorus. a, and 8, fractions Were

-similar in amino acid composition while all and p, were different in histi- .dine, isoleucine and half-cystine.

Clzemistry o f High Density Lipoproteins 143

Amphibian egg yolk lipovifellins

Yolk proteim of a few reptiler and fishes have been purified and ch.arac- terised by TEAE-cellulose ~h~omatography but their properties have not been reported [I 161. Frog egg yolk platelets exist jn a crystirlline state and each platelet is made of two moles of phosvjtin and one mole of ljpo- vitellin [112, 1131. LipoviteIlin-s of frog has only one component which contains 17.5% lipid, 81.5% protein m d 0.45% p~otein phospb.orus. i t has a molecular weight of 430,000. Amino acid composition of frog apovitellin is similar to that of hen.'s egg yolk apovitcllins with the exccptior of ag i - nine and proline. Apovitellins of Xenopus laevis consists of two proteins in the ratio of 1 : 1. The molecular wtigh.t of the two proteins are 31,000 and 120,000. Thc smeller submit h.as 2% phosphorus wl?.iIe the 11ee.vier subunit does not contain phosphorus.

Teleostean lipovitellins

Lipovitellins have been isolated from 13 species of teleosts [I161 and they have been fractionated into a- and /3-lipo~ltellin,. B-lipoviiellins occur in trace: or totally absent in species like alewife, mudmin~.ow, piperfish, sheepshed minnow, iilver sides and butter fish.. However, it is a major component in crappiei sauger and toad fish. Phosvitir? is absent in m a ~ y species. Lipid content of lipovitellins range from 15-29%. A feature of these lipov-tellins is that C-lipovitellin is less phosphorylated than B-Lv. For example in toad fish, a- and P-lipovitellins contain 0.35% and 0.56% protein phosphorus respectively.

Crustacean lipovitellins

Several investigators have studied the lipovitellins of crustaceans. Wallace et a1 have studied the lipovitellins of six decapods ~1171. They have an average molecular weight of 350,000 with a relatively large lipid content of 30%. Protein does not contain phosphorus. These 1ipovitellin.s do not undergo dissociatioa a t alkaline pH but exhibit m inter-se and vilriable absorptivity in th.e visible spectra. Tbme factors mzy be influenced by diet or cnviron.mental conditions. In wn tps t t o decapod lipovitellin.~, an an.ostracm lipovitellin isolated from Branchipus stagnalis exhibited a slow and reversible association-dissociatior. pb.er.omencm :

[121]. Dimexisation was obsel.ved at higher ior.ic strength. The.moleculi?r ;

weight of an anostracan lipovitellin is 630,000. It contains 4 molecules

of canthaxanthin as the caratenoid component and about 15% phospholipid ~f total dry weight, SDS-gel pattern of lipovitellin shows at least five.major

1 44 D. N. RAMAKRISHNA RAO AND S . MAIIADEVAN

bands [121]. k n i n o ricid comnpositioll. of anostrncan lipovi(ellin is closely related to the arnin.0 acid composition or decnpods mid olher. i17,vertebrates, ~ i p ~ ~ i t e l l i n s obtained fronl Procmzbrim species (any fish,) have a male- cubar wejgl~t of 500,000 [IZO]. It contains 35% lipid an.cl it coilsists of a carotenoid, phosphatidyl cholir~c and phosph.atidy1 ethri~~olami~.~. ~~~~~~i~ and glutarnic acids are the lll:+i' ~111~110 acids of the protein. ' Tt exhjbi[s a p~~-dependcnt spectral shifts. Ai p'M' 7 it appC:Lr.s brown wl~ile at pH 8 it appears ol-mge. Th.e differelm in the visible spectrum at these two p~ ,Ire reversible [120]. In. ihe cnsc of 1n:xiil.c inveclebsatbs like coneEr p g u r w and Pecten nm.uinzus the lipovilclli~?.~ contiiin abol~t 27%.lipid. of Mrhich two-thirds is phospholipids and 20-25% cholcsierol [I 181. he mole- cular weight of lipovitcllins in tJ1.e rout+ speci.es qtudied rmges fi-om 170,000- 630,000. Mannose i s the majm sl1g:n- con,;isling 2 .5% of pi'oteill by diy whght. Cholesteryl ester is abst.111 [I 181.

Zaglasky has studied the an1in.o acid cornposition of various cixstacean lipovitcllins [I 191. All of then1 11,ave 3 high proportio~?. of ylutamjc and. aspartic acids. Lipovitellins OF Wonzuntr grrmcirlts arzd Plesionfku ed&rdsi do not contain cysteine or cystine. Thc iiinilaritics in con~position become more apparent when. the con te~~ t s of groups of arni1.0 acids and their ratios consideied. The values for the nverage hydrophobicily and percentage of non. p o l ~ r a1ni.n.o acid.s are similar to thosc found .in globular proteins.

is unusual to h u e a h.iyh content of hclix brcnking unino acidslike swine, threonine and proline. The low crysteine and helical content indicates random coil configuraiion for these proteins [I 191. . . ,

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