Isolation and characterization of a cholesterol crystallization promoter from human bile

Isolation and Characterization of a Cholesterol Crystallization Promoter From Human Bile MASATO ABEI, PAUL KAWCZAK, and R. THOMAS HOLZBACH Gastrointestinal

HANNU

NUUTINEN,

Research Unit, Research Institute and Department

upersaturation

in bile is a necessary

of cholesterol

but not sufficient

condition

for the formation

of

cholesterol gallstone disease.‘,2 Thus, the importance of kinetic factors capable of affecting the rate at which cholesterol

crystallization

occurs

has

been

postu-

lated.3*4 A crystal-forming or growth rate inhibitor,5-7 for example, might retard the process to such a degree that other factors, such as gallbladder contraction, could eliminate the submicroscopic crystals fast enough to prevent crystal growth. Evidence for the existence of such a cholesterol-crystallization inhibitor in the form of a specific glycoprotein has recently been provided.’ The opposing

side of the kinetic

equation

concerns

LANGNAS,

factors

JOAR SVANVIK,

Cleveland Clinic Foundation, Cleveland, Ohio

of Gastroenterology,

Background: Recent studies on the pathogenesis of cholesterol gallstone disease have focused on the potential importance of an imbalance between biliary proteins having either inhibitory or promoting activities on nucleation and/or growth of cholesterol crystals as the initial stage in stone formation. The current study describes the purification and partial characterization of a 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. Methods: Chromatographic methods were used for separation and purification. Characterization steps included electrophoresis, deglycosylation, amino acid and carbohydrate analysis, and activity analysis by crystal growth assay. Results: The 42-kilodalton purified glycoprotein is an extensively glycosylated (37%) monomer with an acidic isoelectric point (PI< 4.1) that is probably based on the sialic acid content of the carbohydrate moiety. Enzymatic N-deglycosylation removes the carbohydrate moiety and inactivates the promoting activity. Furthermore, enzymatic proteolysis results in both its complete structural degradation and functional inactivation. Although the glycoprotein was isolated from normal human gallbladder biles, its presence in gallstone-associated samples is clearly shown. Conclusions: This report outlines biochemical features of a human biliary glycoprotein that may be of major pathophysiological significance in gallstone disease.

S

ALAN

that promote

terol

crystallization

or accelerate

the rate of choles-

in supersaturated

bile.8-”

Mucus

glycoprotein has been reported as an example of such a promoter, but its role in the pathogenesis of gallstone disease dence

is still controversial.‘0-‘2 for the existence

the concanavalin man

bile

A (con

has been

More

of crystallization A)-binding

provided.lS”

recently, fraction

The

evi-

promoters

in

of hu-

promoters

re-

cently identified from this con A-bound fraction are an as yet uncharacterized 130-kilodalton glycoprotein18 and some high-molecular-weight, polymeric immunoglobulins recent

(IgM and probably

IgA).‘” This

most

work 8,9 by the same group also report significant promoting activity by a lower molecular weight (< 1 10-kilodalton) fraction. The explanation clear.

study”

and earlier

for this activity

remains

unspecified

and un-

We describe the purification and considerable characterization of a different 42-kilodalton promoter glycoprotein, which was isolated using reverse-phase high performance liquid chromatography (HPLC) and a cholesterol crystal growth assay.20 The glycoprotein is an extensively glycosylated (37%) monomer containing a high number of sialic acid residues in the carbohydrate moiety, which probably explains its comparatively acidic isoelectric point (PI < 4.1). Further, N-deglycosylation inactivates the promoting activity and enzymatic proteolysis results in both its structural degradation and functional inactivation. Immunochemical studies uniformly failed to show any relationships between the 42-kilodalton glycoprotein and the biliary immunoglobulins or their subunit fragments. Abbreviations used in this paper: AAG, a,-acid glycoprotein; BAPL, bile acid-phospholipid ratio; CSI, cholesterol saturation index; CABG, con A-bound glycoprotein; GS/MS, gas chromatography/ mass spectrometry; I,, nucleation time index; I,, growth index; pl, isoelectric points; HPLC, high-performance liquid chromatography; slgA, secretory IgA; STC, sodium taurocholic acid; STDC, sodium taurodeoxycholic acid; TBS, Tris buffered saline; TL, total lipid concentration; TFA, trifluoroacetic acid. 0 1993 by the American Gastroenterological Association OOIS-~OSS/93/$3.00

540

ABEI ET AL.

GASTROENTEROLOGY Vol. 104, No. 2

Materials and Methods

of the control

Bile Collection

the effect on the maximum the experimental

The protocol was approved by the Cleveland Clinic Foundation’s Research Projects and Institutional Review Committee were

regarding

collected

gallbladder plant

human

during

studies.

surgery

or from cadaver

programs

10 separate

from

identified

bile samples

patients

with

by use of previously

ria.4 These gallbladder to 4 months. the thawed

aspiration

described.”

aspirates

of the

These

patients.

samples

In addition,

were obtained cholesterol

defined

at chole-

morphological

were not observed

for up

in any of

bile samples.

Bile acid concentrations cally.‘i

Lecithin

method

of Bartlett,**

were

concentrations

and cholesterol

assayed enzymatically

using

kit (Boehringer-Mannheim tein concentrations

measured

were

enzymati-

available

Corp., Indianapolis,

priate

aliquots

Kodak

Co.,

Products,

of stock

taurocholic

Nutfield,

terol saturation ratio (BA-PL)

HC1/150

bile were

37°C with shaking.

NaCl

with

either

protein

samples

The cholesterol

by sampling

and diluting

crystal growth

model bile without

(control)

incubated

crystal concentration

monitored

with

sodium taurodeoxycholic acid (STDC)/TBS tion for 20 minutes, followed by absorbance 900 nm. The cholesterol

7.4 at 55’C. (50 pL)

column NaCl,

pH 7.45. Fractions

molecular (fraction

weight 1) that

outlined

A-binding mucin.

other biliary

from kilodal-

glycoproteins.

The

study for removing

investigators

mucin

glycoprowho have stud-

glycoproteins our results

affinity

without

first re-

may not be entirely

mol/L

(starting

was dialyzed

NaCl,

MnCl,, buffer)

(Amicon,

sion limit,

chromatography.

weight)

1 mmol/L

Sepharose

Beverly,

Inc.,

0.2 mol/L

4 mmol/L

ultrafiltration

MA) with a YM 10 filter (excluit was then NJ), which

to remove

bound

applied

to a con A

was extensively nonadsorbed

to the column

weight)

as CABG

materials. eluted

with with The with

(Sigma Chemical

buffer.

The con A-bound fraction

(hereafter

was concentrated

in

ity on the cholesterol crystal growth assay. Reverse-phase HPLC. An aliquot of CABG fraction was initially incubated in 80% acetonitrile/O. 1% trifluoro-

curves of the superand with (experimen-

from the experimental curves to those from the control curves.” The nucleation time index (If) represents the effect of the protein samples on the onset time of crystal detection by onset time

showed

acetic acid (TFA)

ammonium

bicarbonate,

and dia-

This fraction

10 mmol/L

25 mmol/L

washed

were then

glycoprotein

fraction)

Biotech-

was equilibrated

a-D-methyl-mannopyranoside

molecular

1 mmol/L

STC, and 1.5 mmol/

by using an Amicon

The column

buffer

designated

10 mmol/L

CaCl,,

(1.6 X 30 cm; LKB/Pharmacia

Piscataway,

glycoproteins

The fraction

with

1 mmol/L

10 kilodaltons); column

the same buffer.

(lower

(>200

to theirs.“,”

Tris HCb’O.5

nology

mucus

kilodal-

separated

high-molecular-weight

Therefore

molecular

L NaN,

included

(<200

were

fractions

in the present

and

moving

lipids

protein

from that of other

ied con

MgCl,,

proteins

of the biliary

at

an ali-

4

were

lyzed against

(475 FL) solumeasurement at

curve divided

The

mmol/L

or

tal) protein samples were thus generated for each sample.” Two activity indices were calculated as the ratio of values

(onset time of the experimental

previously.”

Tris HCl/150

Co., St. Louis, MO) in the starting

(325 PL) of this model

(50 FL of TBS) and were

was sequentially

to

with 25 mmol/ pH

most

the starting

molar

was evaporated

(TBS),

aliquots

quot (25 FL) of the mixture

saturated

CA) were

model bile with a choles-

and then resolubilized mmol/L

solutions

the mixture

San Diego,

sodium

and a bile acid-phospholipid

(0.22 pm),

mixed

I; Lipid

and

CA) to

as well as high-molecu-

low-molecular-weight

and

procedure

system

(Eastman

(grade

England),

of 4.4. This lipid mixture

lyophilized,

After filtration control

Surrey,

by

appro-

(CSI) of 1.4, a total lipid concentra-

tion (TL) of 12.5 g/dL,

L Tris

of cholesterol

a supersaturated index

were assessed

egg lecithin

acid (STC) (Calbiochem,

mixed to construct

dryness,

solutions NY),

as described

Richmond,

bile col-

for protein,24325 and two well-defined peaks were obtained. The main protein peak (fraction 2)

2 (lower

assay.17s20 Briefly,

Laboratories,

10 mmol/L

Con A-lectin crystal-

of the

reproducibly

compatible

on cholesterol

kinetics

photometric

Rochester,

South

assayed

glycoproteins

protein

slope of

slope

Procedures

STC, and 0.02% NaN,,

teins differs

IN).23 Pro-

by fluorometric

samples

and growth)

described

mmol/L

were

acid precipitation.25

The effects of protein (nucleation

protein

assay

Cholesterol Crystal Growth Assay

previously

lar-weight

tons)

by the

(Ir) represents

by maximal

soluble mucus glycoproteins

higher

concentrations

a commercially

were measured

assayz4 with trichloroacetic

lization

umn (5 X 100 cm; Bio-Rad

tons)

determined

divided

index

rate (maximum

Gel filtration chromatography. Pooled normal specimens were initially separated by a Bio-Gel A-5M

containing

Lipid and Protein Assay Procedures

curve

growth

Protein Purification

was run with

crite-

and the growth

curve).

remove

cholelithiasis

were stored at -80°C

Cryoprecipitates

control

bile samples

in liver or kidney trans-

to be from stone-free

gallbladder

cystectomy

Human

by needle

donors

as previously

were considered

curve),

pH 8.

strongcrystallization-promotingactiv-

(Pierce

Chemical

Co., Rockford,

IL) for 5

hours, followed by dialysis with TBS. The cholesterol crystallization-promoting activity of the acetonitrile-treated CABG was then compared with a comparable aliquot of the untreated CABG fraction. Treatment with organic solvent failed to alter the promoting activity of the CABG fraction. On the basis of this finding, it was felt reasonable to preceed with attempts to further separate the CABG fraction by use of a reverse-phase HPLC column (Vydac Separation Group, Hesperia, CA) attached to an HPLC system (model 550E

CHOLESTEROL CRYSTALLIZATION PROMOTER IN BILE

February 1993

HPLC; Waters Chromatography

Division,

Millipore Corp.,

541

chains as well as light chains of human IgG, IgA, and IgM,

Milford, MA).‘“** Separation was achieved by using a gradient formed by 0.1% TFA in Ha0 (buffer A) and 0.1% TFA

was coupled to an AminoLink

in acetonitrile

aldehydes that react with the primary amine groups of anti-

(buffer B). After equilibrating

the initial buffer concentrations

the column at

(buffer A, 75%; buffer B,

manufacturer’s

specifications.

gel (Pierce)

according

The AminoLink

bodies and thus can form a stable covalent

linkage with

25%), the sample was applied. The column was then eluted

minimal

with a shallow

fraction (5 mg) was applied to the column, which was equili-

25%55%

linear gradient

in 150 minutes).

of acetonitrile

(buffer

Flow rate was maintained

B, at 1

mL/min, and absorbance was monitored at a wavelength of

brated

leakage of immobilized

to

gel contains

with

25 mmol/L

Tris

antibodies. HCl/150

The CABG

mmol/L

NaCl

(TBS), pH 7.4. The column was allowed to equilibrate over-

280 nm. Fractions of 1 mL were collected in tubes contain-

night at 4°C to completely

ing 25 mmol/L ammonium

the applied sample. The unbound materials were examined

bicarbonate,

pH 8, which im-

remove immunoglobulin

from

mediately neutralized the elution buffer pH, followed by

by SDS-PAGE

dialysis with TBS.

the unbound fraction still contained bands corresponding

phoresis (SDS-PAGE)

(4%20%

then re-loaded onto the affinity column. This procedure was

gel electro-

gradient) was performed

repeated as necessary until a completely

polyacrylamide (Ampholine

focusing

(IEF)

gel containing

PAG

was performed

power of 20 W for 2 hours. After fixation,

was deglycosylated

the gels were

proaches (N-deglycosylation

blot analysis 3’ for immunoglobulin

formed using either rabbit polyclonal polyvalent generic immunoglobulins ies to individual anti-&G

(y chain), and anti-secretory

membrane

(l,t chain),

(sIg)A (a chain plus

which were obtained

and Boehringer-Mannheim. SDS-PAGE

antibody to human [anti-IgM

from Sigma

First, the proteins separated by

were transferred to a polyvinylidene (Immobilon

in 48 mmol/L Tris, 39 mmol/L nol, pH 9.2, using a Trans-Blot retie transfer

difluoride

P; Millipore Corp., Bedford,

MA)

glycine, and 20% metha-

SD semidry blot electropho-

system (Bio-Rad).

Electrophoretic

using

two

promoter different

and 0-deglycosylation)

transfer

nent,

the

type

carbohydrate

of

linkage

components,

between

polypeptide

using

N-glycanase

Corp., Boston,

enzyme

(N-glycosidase

MA) was performed

plier’s specifications.

The glycoprotein

with sodium phosphate

sample was diluted

1 ,lO-phenanthroline

and was incubated with iV-glycanase

(60 U/mL) at 37°C.

After 72 hours, the mixture was dia-

lyzed against 25 mmol/L ammonium examined on SDS-PAGE

bicarbonate

growth assay. In the 0-deglycosylation

incubated with 1 U/mL of Neuraminidase hydrolase; Genzyme)

incubated in anti-human

lowed by further incubation

ZO/TBS between each incubation.

Finally, color was devel-

crystal

study, the glycoprocalcium acetate

sodium cacodylate buffer (pH 7) and was

150 mmol/L NaCl, pH 7.4 (TBS). The membrane was then

body solution (Sigma), with extensive washing in 5% Tween

and then

as well as on cholesterol

with 3% bovine serum albumin in 25 mmol/L

secondary anti-

Genzyme to the sup-

buffer (pH 8.6) and 10 mmol/L

tein sample was diluted with 10 mmol/L

Ig antibody solution followed by

F,

according

and 20 mmol/L

Tris HCI/

and

on the functional activity of the protein, N-Deglycosylation

After the transfer, the membrane was blocked by incubation

in alkaline-phosphatase-labeled

ap-

to de-

and the effect of deglycosylation

was completed in 36 minutes at a constant voltage of 20 V.

incubation

glycoprotein

enzymatic

termine the molecular weight solely of the protein compo-

was per-

or polyclonal antibod-

immunoglobulins

secretory component)],

The purified 42-kilodalton

at a constant

stained with silver nitrate by the method of Morrissey.30 Western

Deglycosylation Studies

in a 5%

5% ampholyte (pH 3.5-9.5)

plate; LKB/Pharmacia)

immunoglobulin-

free fraction was obtained.

using the standard techniques described by Laemmli.29 Analytical isoelectric

to

either IgM, IgG or IgA, by either method, this fraction was

Electrophoresis and Western Blotting Sodium dodecyl sulfate-polyacrylamide

stained with silver and by Western blot. If

(acylneuraminyl

for 12 hours at 37°C.

acetylgalactosaminidase;

This was fol-

with 0-glycanase

Genzyme)

(endo-a-N-

for 72 hours at 37°C.

Finally, the mixture was dialyzed against 25 mmol/L ammonium bicarbonate

and examined on SDS-PAGE.

For con-

oped by exposing the membrane to 154 mmol/L 5-bromo-

trol purposes,

4-chloro-3-indolyl

chemicals or enzymes as indicated above but not containing

phosphate, 77 mmol/L nitroblue tetrazo-

lium in 100 mmol/L mmol/L

MgCl,,

Tris HCl, 100 mmol/L

NaCl, and 5

pH 9.5.

glycoprotein

the same amount

of

were also incubated (enzyme

Proteolysis Studies

To determine whether the strong cholesterol crystalactivity in the CABG fraction could be

the result of the immunoglobulins reported to possess promoting

containing

controls).

lmmunoabsorption of lmmunoglobulin lization-promoting

the 42-kilodalton

mixtures

that have recently been

activity,”

an anti-human

Ig

antibody column (0.5 X 2 cm) was prepared. Purified goat polyclonal antibody (Sigma, 20 mg) against human generic polyvalent immunoglobulin, which reacts with heavy

Proteolysis was performed as previously described.32 Briefly, 30 pg of purified 42-kilodalton glycoprotein in 25 mmol/L

ammonium

bicarbonate

with 2 U (final concentration,

(pH 9) was incubated

8 U/mL) of Pronase (Strepfo-

mycesgriseus, type XXI; Sigma) at 37’C for 72 hours. As controls, equal amounts of the protein were incubated with equal amounts of Pronase that had been heat-inactivated by

542

ABEI ET AL.

GASTROENTEROLOGY Vol. 104, No. 2

boiling for 5 minutes. Also, incubated. All samples were ammonium bicarbonate and PAGE on cholesterol crystal

Pronase alone (8 U/mL) was dialyzed against 25 mmol/L then examined on both SDSgrowth assay.

Amino Acid Analysis Two samples of the isolated 42-kilodalton glycoprotein were analyzed. The samples were hydrolyzed for 16 hours at 115’C in 6N HC1/0.2% phenol containing norleutine as an internal standard. After this incubation, samples were dried and redissolved in 100 PL of NaS sample dilution buffer (Beckman Instruments Inc., Fullerton, CA) and run on a Beckman model 7300 Amino Acid Analyzer with postcolumn Ninhydrin (Sigma Chemical Co., St. Louis, MO) detection.

Carbohydrate

Analysis

The purified 42-kilodalton glycoprotein was analyzed by preparing trimethylsilyl derivatives of the methyl glycosides, followed by gas chromatography (GC) and combined gas chromatography/mass spectrometry (GC/MS) analysis for neutral and amino sugars. Trimethylsilyl methyl glycosides were prepared by methanolysis in 1 mol/L HCl in methanol, followed by AJ-acetylation with pyridine and acetic anhydride. The sample was then treated with Tri-Sil (Pierce). The procedure was accomplished as previously described.33 GC analysis of the trimethylsilyl methyl glycosides was performed on a Hewlett-Packard 5890 Gas Chromatograph using a Supelco DBl (Supelco, Inc., Bellefonte, PA) fused silica capillary column. GC/MS analysis was performed using a Hewlett-Packard 5890 GC coupled to a

r^ ::

1.0 0 z 0.6 % I= 0.6 6p

0.4

z -I

0.2

Y

A

12345676 FRACTIONS

12345676 FRACTIONS

6 ?? PC005

‘P
Figure 2. Cholesterol crystallization-promoting activity in the reverse-phase HPLC-separated fractions. Each fraction obtained from reverse-phase HPLC (50 pg protein/ml model bile) was mixed with an identical supersaturated model bile solution, and activity indices were determined as described in the text. Significant promoting activity was detected only in fractions 4 and 5 (indicated by dark and shaded bar). Data are given as mean f SD (n = 6; P < 0.05).

5970 Mass Spectrometry Palo Alto, CA).

Detector

(Hewlett-Packard

Co.,

CABG Fraction From Cholesterol Gallstone Patients vs. Stone-Free Subjects To determine whether the 42-kilodalton glycoprotein exists not only in gallstone-free bile but also in gallstone-associated bile, the CABG fraction was obtained from individual gallbladder bile samples using separate small con A Sepharose columns (0.5 X 2.0 cm). Aliquots of gallbladder bile samples from 10 stone-free patients as well as from 10 patients with cholesterol gallstone disease were separately applied to the columns, which were washed and then eluted as described above. The CABG fraction from each sample was concentrated, dialyzed against 25 mmol/L ammonium bicarbonate, and then run on SDS-PAGE followed by silver stain.

Statistical

Methods

Calculation of standard deviations in crystal growth curves data was performed by application of the standard Pearson method (root-mean-square deviation). Comparative analysis was performed using Student’s t test.

Results I

20

40

I

I 100 120 60 ELUTION VOLUME (ml)

Purification of a Cholesterol Crystallization-Promoting Glycoprotein

I

140

160

Figure 1. Separation of CABG by reverse-phase HPLC. The CABG fraction that contains strong cholesterol crystallization-promoting activity was applied to a reverse-phase HPLC column (0.46 X 25 cm). The column was eluted using a gradient formed by 0.1% TFA in H,O (buffer A) and 0.1% TFA in acetonitrile (buffer B). The sample was separated into eight fractions. Only fractions 4 and 5 (indicated by dark and shaded peaks) showed detectable promoting activity as shown in Figure 2.

The CABG fraction was separated into eight subfractions by reverse-phase HPLC (Figure 1). These fractions (50 pg protein/ml model bile) were mixed with a supersaturated model bile solution (CSI, 1.4; TL, 12.5 g/dL; BA-PL, 4.4), and the cholesterol ctystallization-promoting activity was tested by the cholesterol crystal growth assay as described above. As shown

in Figure

2, significant

promoting

activity

was

CHOLESTEROL CRYSTALLIZATION PROMOTER IN BILE

February 1993

RPHPLCFractions

its subfractions subfractions

CABG

c1

2

3

4

5

6

7

a

were further were

only observed sIgA antibody

analyzed

studied.

anti-h&

antibody

ure 4, lane 11). The fractions crystallization-promoting

antibodies

globulin

Figure 3. SDS-PAGE (reduced conditions) of the CABG fraction and its reverse-phase HPLC-separated subfractions. The only fractions containing promoting activity were fractions 4 and 5 (Figure 2) both of which showed a 42-kilodalton band. This was the only band found in fraction 4, the fraction that showed the greatest promoting activity. Aliquots containing 1 ug of protein were loaded on each lane, and the gel was stained with silver.

fraction

moval

of the

fraction coupled

antibody specific

(Figure

4, lane

not shown).

biliary

showed

no detectable

activity

before

and

immunoglobulin.

blotting

absorption,

the

amounts

(Figure

tions with and without

and the eight subfractions HPLC separation. promoting activity 42-kilodalton greater

activity,

result, tein

band,

contains

moting

from reverse-phase

4, which

only this band.

shows

Based on this

that the 42-kilodalton

be responsible

the

glycopro-

The

The CABG fraction blotting using anti-human (Figure

reported

CABG

CABG

fraction

of immunoglobulin

immunoglobulin

model 4.4),

bile solution and

the

(CSI,

effect

h+TX”;” 1 2 3

4

on

on frac-

(100 pg/rnL

1.4; TL,

supersat12.5 g/dL,

cholesterol

crystal

Anti-slgA Anti- Anti-

5

6

7 a

9

10 11 12

205106Bo-

Western Blotting and lmmunoabsorption of lmmunoglobulins

ously

re-

for the crystallization-pro-

activity.

antibody

(2)

of the

after

4, lane 5). The CABG

Anti-19

subfractions that show 4 and 5) both contain a

and fraction

we concluded must

obtained

The two (fractions

BA-PL,

6) or

immuno-

model bile) were then mixed with an identical detected exclusively in fractions 4 and 5 (P < 0.05). Figure 3 shows the SDS-PAGE pattern of the CABG

2 (frac-

was applied to an immunoabsorption column with the anti-human generic immunoglobulin After

urated

8 (Fig-

the generic

to each individual

was compared

antibody. Western

either

the crystallization-promoting

CABG

the

cholesterol

in Figure

to react with

class (results

Finally,

in fraction

that showed

activity

4 and 5) failed

anti-immunoglobulin with

the eight

blotting,

positive reactions were against antiin fractions 6 and 7 (Figure 4, lanes 8

and 9) and against

tions

When

on Western

543

was analyzed by Western generic immunoglobulin

4, lane 4), which presence

confirmed

of immunoglobulin

and sIgA], the CABG with anti-sIgA, slight

in the

fraction staining

with anti-&$, and no reaction with anti-IgM (Figure 4, lanes 7, 10, and 12, respectively). These findings indicate that the CABG fraction contains a considerable amount of sIgA, trace amounts of IgG, but no detectable amount of IgM. To presence of Ig could contribute ity of the CABG fraction and HPLC-separated subfractions,

;;r 19-

the previ-

CABG fraction. l9 On Western blotting, using antibodies specific to individual immunoglobulins [IgM(p chain), IgG (‘y chain), showed strong staining

5C-

determine whether the to the promoting activalso of its reverse-phase the CABG fraction and

Figure 4. lmmunoglobulins present in the CABG fraction and in its reverse-phase HPLC-separated subfractions as analyzed by Western blotting. Western blot analysis using anti-immunoglobulin showed the presence of immunoglobulin in the CABG fraction (lane 4) and the absence of immunoglobulin in the CABG fraction after immunoabsorption of immunoglobulin (lane 5). Further analysis of the CABG fraction, using antibodies specific to slgA, IgG, or IgM, showed strong staining with anti-slgA (lane 7), slight staining with anti-IgG (lane IO), and no staining with anti-IgM (lane 12). Among the reverse-phase HPLC-separated subfractions, the only positive immunoreactivity observed was in fractions 6 and 7 against anti-slgA (lanes 8 and 9) and in fraction 8 against anti-IgG (lane 11). Fraction 4, which contains only the 42-kilodalton glycoprotein, did not react with anti-immunoglobulin antibody (lane 6). Lane 1, IgG standard: lane 2, IgA standard; lane 3, IgM standard; lanes 4, 7, 10, and 12, CABG fraction: lane 5, CABG fraction after absorption of biliary Ig: lane 6, fraction 4 (pure 42-kilodalton glycoprotein); lane 8, fraction 6; lane 9, fraction 7; lane 11, fraction 8.

544

ABEI ET AL.

GASTROENTEROLOGY

in

the

band

disappearance and

the

of the

appearance

band on SDS-PAGE assays performed lesterol

original

of a strong

(Figure

Vol. 104.

No. 2

42-kilodalton 26-kilodalton

8). On the crystal growth

after the N-deglycosylation,

crystallization-promoting

activity

no chowas detected

(Table

1). Th’is a b sence indicates that the carbohydrate moiety is essential to the promoting activity of this protein. Treatment with no effect on SDS-PAGE; 20

0

40

60

60 TIME

100

120

140

160

160

band

(hours)

Figure 5. Comparison of the cholesterol crystallization-promoting effect of the CABG fractions with and without absorption of immunoglobulins. The CABG fractions (100 pg protein/ml model bile) with and without immunoabsorption of immunoglobulin were mixed with an identical supersaturated model bile solution as described in the text (control). The immunoabsorption removal of immunoglobulin did not alter the promoting effect of the CABG fraction. The control curve (n = 5) and the experimental curves (n = 4) are given as mean f SD. - 0 -, Control (n = 5); - - A - -, CABG (n = 4); - - 0 - -, CABG - Igs (n = 4).

growth moting

was compared. As shown in Figure 5, the proactivity of the CABG fraction was not altered

by the presence

or absence

was unaltered

enzyme

of immunoglobulin.

These

the CABG

crystallization-promoting fraction

moter glycoprotein

and

that

activity the 42-kilodalton

is not an immunoglobulin

found

terol

crystallization

Proteolysis Pronase in the

Characterization Glycoprotein

of the Purified Promoter

model

ton glycoprotein were mixed with bile solution shown

bile) of the purified

(reverse-phase an identical 6, the

42-kilodal-

HPLC fraction 4) supersaturated model

(CSI, 1.4; TL, 12.5 g/dL;

in Figure

None

lacked

of the

the 42-ki-

crystal

growth

as-

studies.

promoting coprotein

The

proteolytic

effect

of

glycoprotein is represented Figure 8. All incubations

active Pronase on SDS-PAGE.

showed complete protein As shown in Table 1, no

activity was detected with the purified glyafter proteolysis. With inactivated Pronase,

no digestion shown).

was observed

on SDS-PAGE

(results

not

4’oL 3.0 8 8 ‘:

2.0 -

% l.O-

Concentration dependence of crystallization(25, 50, promoting activity. Different concentrations and 100 &q’mL

(which

by cholesterol

on the promoter right lane of

containing degradation

pro-

nent.

not shown).

say.

in

compo-

(results

experiments

lodalton glycoprotein) showed stainable protein bands on SDS-PAGE, and none showed an effect on choles-

findings indicate that neither the biliary immunoglobulin nor their derived fragments are responsible for the cholesterol

control

0-glycanase enzyme showed the original 42-kilodalton

42-kilodalton

BA-PL,

4.4). As

glycoprotein

shows a definite concentration-dependent promoting effect on the cholesterol crystal growth curve. SDS-PAGE and IEF. As shown in Figure 7, the purified glycoprotein showed a single band of 42-kilodalton on SDS-PAGE under both nonreduced and reduced conditions. On the other hand, multiple bands were seen on IEF, all having an acidic pI(<4.1). These findings indicate that the glycoprotein is an acidic monomer with considerable microheterogeneity (isoforms). Deglycosylation studies. Treating the 42-kilodalton glycoprotein with N-glycanase enzyme resulted

0.0

L

0

20

40

60

80

100 120 140 160 180

TIME (hours) Figure 6. Concentration dependence of the promoting effect of the 42-kilodalton glycoprotein on the cholesterol crystal growth curve. Three concentrations of the purified 42-kilodalton glycoprotein (25, 50, and 100 @JmL model bile) were mixed with an identical supersaturated model bile solution as described in the text. The 42-kilodalton glycoprotein showed a concentration-dependent promoting effect on the crystal growth curve. The control curve is given as mean + SD (n = 5). Each of the experimental curves is given as mean (n = 2). Selection of the concentration range used in these studies was based on the following considerations: a known amount of purified 42-kilodalton promoter was taken through the entire procedure with an estimated “recovery” of 20%. Radioactive labeling of a smaller amount of 42-kilodalton promoter and addition of it to the starting material led to a “recovery” estimate again in the same range asforthe unlabeled material. From calculations involving estimates of the starting amount of total biliary protein including several reasonable assumptions, it was estimated that the original gallbladder bile concentration for the 42-kilodalton glycoprotein should be at least in the range of at least 50 pg/mL. - 0 -, Control (n = 5); - - A - -, 25 pg/mL (n = 2); -~O~-,50~g/mL(n=2);--U----, lOOpg/mL(n=2).

CHOLESTEROL CRYSTALLIZATION PROMOTER IN BILE

February 1993

A

Table 1. Effect of the 42-Kilodalton

Glycoprotein on Activity Indices of Cholesterol Crystallization

B

12

545

PI

.i

Activity indices Concentratron @g/ml)

+ N-deglycosylation + Proteolysis “Within the uncertainty

Non-Reduced 2: Reduced 1:

by relatively

amino

acids:

moiety Figure 7. SDS-PAGE (reduced and nonreduced conditions) (A) and IEF (8) of the purified promoter glycoprotein. The glycoprotein showed a single 42-kilodalton band on SDS-PAGE under both reduced and nonreduced conditions. On the other hand, multiple bands were seen on IEF, each having an acidic pl (14.1). The gels were stained with silver.

the purified

promoter

deglycosylated is composed

portion

Amino

glycoprotein

acid

showed

(26 kilodaltons)

of 199 amino

analysis

acid residues

that

of the

of the protein and is charac-

of the following acid,

(Table

of the glycoprotein

asparagine/

2).

analysis.

The

(37%)

carbohydrate

is almost

entirely

of only four monosaccharide

i.e., N-acetylglucosamine, galactose in nearly equal

species,

sialic acid, mannose, amounts (Table 3).

and

CABG Fraction From Patients With Cholesterol Gallstones vs. Stone-Free Subjects Figure the respective samples

9 compares the SDS-PAGE pattern of CABG fractions from five separate bile

obtained

from

stones and three stone-free patients. ined, PAGE

patients

band

as one of the prominent shows

glycoprotein

with

cholesterol

gall-

separate samples obtained In each of the 20 samples

the 42-kilodalton

this band

Mr W)

amounts

glutamine/glutamic

acid, and leucine

(97%) comprised

Amino acid analysis.

large

Carbohydrate

6.0 -

lg 1.29 1.62 2.53 I .08’ 1.12”

1.oo* 0.75 0.64 0.94* 1.05*

range of control measurements.

terized aspartic

It

25 50 100 50 50

Purified 42-kilodalton

qualitatively

exists

not only

was observed bands. that

from exam-

on SDS-

The presence

of

the 42-kilodalton

in gallbladder

bile from

200 ‘F

-

Table 2. Amino Acid Composition of the 42-Kilodalton Promoter Glycoprotein

66 45 -

4

Amino acid Glx Asx Leu

31 -

4 Front Promotor Glycoprotein

Enzymatic N-Deglycosylation

Proteolytic Digestion

Figure 8. SDS-PAGE (reduced conditions) of the purified 42-kilodalton glycoprotein after N-deglycosylation and after proteolysis. A single 26-kilodalton polypeptide band was obtained on SDS-PAGE afterenzymatic N-deglycosylation of the pure 42-kilodalton glycoprotein. Complete digestion was observed after treatment with Pronase (Streptomyces griseus) at 37°C for 72 hours. The gels were stained with silver.

LYS Gly Thr Ser Val Ala Pro Arg Tyr lleu Phe His Met Total

No. of residues

Mole percent

30.3 23.5 16.0 14.8 14.1 12.6 12.0 11.9 11.7 10.7 9.5 9.3 8.4 6.7 5.6 1.4 198.5

15.2 11.8 8.1 7.5 7.1 6.4 6.0 6.0 5.9 5.4 4.8 4.7 4.2 3.4 2.8 0.7 100.0

546

ABEI ET AL.

GASTROENTEROLOGY

Table 3. Carbohydrate

Composition Promoter Glycoprotein

Glycosyl residue=

of the 42-Kilodalton

resistant

Percent*

Mass 018,

29.5 26.1 19.6 19.6 2.3 1.5 1.3 ND 100

27.1 24.0 18.0 18.0 2.1 1.4 1.2 ND 91.8

N-acetylglucosamine Sialic acid Mannose Galactose Glucose Fucose Xylose GalNac Total

130-kilodalton

CABG have

fraction

also identified

A-bound

the

which

was included

tion

Because

the 130-kilodalton

remained

uncharacterized

we are unable

but also in bile from

patients

resolution tain

Discussion

related The idea for a protein-related thoughtfully

conceived

by Burnstein explain

effect

a 130-kilodalton

with

greater

potency

most

recent

work

biliary

and were

into three fractions, was observed

intact

polymeric

fraction,

the cause

lowest molecular from their report. fied and partially

molecular et al.

et al. has

of low purification

compare

their

results

was based

polypeptide variety

tions,

successful

ously

described

from

of other

Despite

solvents

exposure

during

reverse-phase

of

separation

the

CABG

about

HPLC

IgM

dence

of functional

of IgA.‘” In this

partial

denaturation

and

proteins

HPLC

to

separa-

has been previ-

of studies without functhe present study, the

approach fraction

provided without

impairment. and

to obfraction

the denatur-

of certain

reverse-phase

use of this approach

of the CABG

on pre-

chromatographic

concerns

in a variety 26-28 In impairment.

group

as a high-

investigators

ing effect of transient

to

HPLC

consequent

The

satisfactory obvious possibility

partial

eviof

loss of

A-bound

by gel filtration promoting

the highest

molecular

activity weight

and in the lowest molecukilodaltons). Whereas the

immunoglobulin activity

our

of Groen

of Groen

because

to directly

purification

methods.

organic

tional

similar

protein

work

perhaps

tool

a wide

binds

of biliary

et a1.,19 con

separated

and a definite

in both

fraction (>500 kilodaltons) lar weight fraction (
possibly

to

glycopro-

that

primarily

by Harvey

glycoproteins

reported

a con A-binding

consisting

the

thought

mucin

glycoprotein

con A,18 and most recently large glycoproteins

Proteins

included

crys-

with

experiments

1983.8

have

cholesterol

originated

mixing

et al. in

this

teins,%”

activity

previous

as that

no

the isola-

by both us and other

a single

using

tallization-promoting

nearly

use of reverse-phase

final

vious failure

disease.

However,

ours.

Our empirical gallstone

observed,

in our later

Therefore,

may not be the same

yield,

others

(7 and 8), showed

ours. of

we con

molecular we

et al. in their

from

glycoprotein

weight

with patients

several activity.

of Green

strikingly

from the

in the crude

approximate

fractions

No. 2

et al. l8 Whereas

glycoprotein with

HPLC

method

differed inactive

gallstone-free

this

crystallization-promoting

ND, not detected. %ugar composition for both the neutral and amino sugars were determined by the trimethylsilyl method. bValues are expressed as the percent of total carbohydrate. Thirtyseven percent of the mass of the sample is carbohydrate.

with cholesterol

of

particular

reverse-phase

isolated

by Groen

a glycoprotein

fraction

weight,

glycoprotein

reported

Vol. 104,

could easily explain

of the highest

molecular

of the promoting

activity

weight in the

weight fraction remains unclear The 42-kilodalton glycoprotein puricharacterized in the present report

4 42

could explain the low-molecular-weight promoting activity noted in their study. In light of their convincing evidence

for immunoglobulin-related

promoting

activ-

ity, we took considerable care to exclude the possibility that the present glycoprotein is somehow derived from, or shares immunochemical features with, the immunoglobulin family of glycoproteins. Another

potential

candidate

promoter

is a Pronase-

Figure 9. The presence of the 42-kilodalton glycoprotein in CABG fractions from normal and cholesterol gallstone-associated biles. CABG fractions were obtained from individual bile samples and run on SDS-PAGE. In every sample, a 42-kilodalton band was observed on SDS-PAGE as one of the prominent bands, regardless of the presence or absence of gallstones. Lanes I-5, con A-bound glycoproteins from stone-associated gallbladder biles; lanes 6-8, con A-bound glycoproteins from stone-free gallbladder biles.

February

CHOLESTEROL

1993

function

cannot

be excluded

until

comparative

po-

accomplished

tency studies become available using the 42-kilodalton promoter isolated by techniques not involving protein

coprotein, involving

exposure to acetonitrile. We showed qualitatively

cance.

coprotein

is present

not

that the 42-kilodalton only

in normal

gly-

we have also found obtained

ease shows tency

that the 42-kilodalton

from patients

with

equivalent

at equivalent

This analysis,

gallstone

dis-

crystallization-promoting

concentration

however,

glycoprotein

cholesterol

data).

relationships.

Quantitative

methods

this described

glycoprotein

must

be devel-

we can only

indicate

that while

the glycoprotein

its pathophysiological biological relevance, cance remains to be clearly established.

3.

Holzbach RT, Busch N. Nucleation and growth of cholesterol crystal: kinetic determinants in supersaturated native bile. Gastroenterol Clin North Am 199 1;20:67-83.

4.

Holan KR, Holzbach RT, Hermann RE. Cooperman AM, Claffey WJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:6 1 l-6 17.

5.

Holzbach RT, Kibe A, Thiel E, Howell JH, Marsh M, Hermann RE. Biliary proteins. Unique inhibitors of cholesterol crystal nucleation in human gallbladder bile. J Clin Invest 1984;73:35-45.

6.

Kibe A, Holzbach RT, LaRusso NF, Mao SJT. Inhibition of cholesterol crystal formation by apolipoproteins in supersaturated model bile. Science 1984;225:5 14-5 16.

7.

Ohya T, Busch N, Egami K, lgimi H, Pillay SP, Takabayashi A, Svanvik J, Holzbach RT. Further studies of a purified glycoprotein from normal human gallbladder bile that inhibits cholesterol crystallization and growth in vitro (abstr). Hepatology 1990; 12:900.

8.

Burnstein MJ, llson RG, Petrunka CN, Strasberg SM. Evidence for a potent nucleation factor in the gallbladder bile of patients with cholesterol gallstones. Gastroenterology 1983;85:80 I-807.

9.

Gallinger S, Harvey PRC, Petrunka CN, llson RG, Strasberg SM. Biliary proteins and the nucleation defect in cholesterol cholelithiasis. Gastroenterology 1987;92:867-875.

10.

Levy PF, Smith BF, LaMont JT. Human gall bladder mucin accelerates nucleation of cholesterol in artificial bile. Gastroenterology 1984;87:270-275.

11.

Gallinger S, Taylor RD, Harvey PRC, Petrunka CN, Strasberg Effect of mucous glycoprotein on nucleation time of human Gastroenterology 1985;89:648-658.

12.

Harvey PRC, Rupar CA, Gallinger S, Petrunka CN, Strasberg SM. Quantitative and qualitative comparison of gallbladder mucus glycoprotein from patients with and without gallstones. Gut 1986;27:374-38 1.

13.

Groen AK, Stout JPJ, Drapers JAG, Hoek FJ, Grijm R, Tytgat GNJ. Cholesterol nucleation-influencing activity in T-tube bile. Hepatology 1988;8:347-352.

14.

Drapers JAG, Groen AK, Stout JPJ, Noordam C, Hoek FJ, Jansen PLM, Tytgat NJ. Quantification of cholesterol nucleation promoting activity in human gallbladder bile. Clin Chim Acta 1987; 165:295-302.

15.

Groen AK, Ottenhoff R, Jansen PLM, van Marle J, Tytgat GNJ. Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile. J Lipid Res 1989;30:5 l-58.

16.

Harvey PRC, Upadhya A, Toth JL, Strasberg SM. Lectin binding characteristics of a cholesterol nucleation promoting protein. Clin Chim Acta 1989;185:185-190.

17.

Busch N, Matiuck N, Sahlin S, Pillay SP, Holzbach RT. Inhibition and promotion of cholesterol crystallization by protein factors from normal human gallbladder bile. J Lipid Res 199 1;32:695702.

18.

Groen AK, Noordam C, Drapers JAG, Egbers P, Jansen PLM, Tytgat GNJ. Isolation of a potent cholesterol nucleation-promoting

has signifi-

Given

the

complexities of this disease process, establishing this role will require a considerable amount of further work. The more striking characteristics of the glycoprotein identified so far relate to carbohydrate composition.

The

protein

comparatively

is heavily

high amount

glycosylated of mannose

(37%).

Its

is not surpris-

ing because of the affinity of the glycoprotein for a con A-lectin column. However, the high preponderance of other monosaccharides in addition to mannose makes it clear that the carbohydrate moiety does not simply

represent

a high-mannose

(oligomannose)

car-

bohydrate chain but more likely a complex-type chain possibly with multiple antennae.34 The sialic acid content, which saccharides,

is the second most abundant of the monois most striking in this respect and, be-

cause of its high concentration,

probably

accounts

for

the comparatively acidic p1 of the glycoprotein. The carbohydrate moiety is essential to the promoting activity of the hologlycoprotein. We have no clear concept of its mode of action in promoting cholesterol crystallization, but some form of interaction with biliary lipid vesicles35 is a likely and testable hypothesis. Recent preliminary studies from this laboratory have provided compelling evidence that the 42-kilodalton glycoprotein is a biliary form of a,-acid glycoprotein (AAG). Th’IS IS b ase d on two findings. The amino acid sequence of a tryptic peptide stretch of 14 amino acids showed a perfect match with that of AAG. In addition, the 42-kilodalton glycoprotein showed immunoreactivity with anti-AAG antibody.36 With the newly

gly-

for studies signifi-

Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile: relationship to gallstone formation and dissolution in man. J Clin Invest 1978;61:988-1026.

can be prop-

erly fitted into the perspective of health and disease. Stated differently, at the present level of information,

of the 42-kilodalton

rapid progress should be possible mechanism and pathophysiological

547

2.

does not deal with quantitative

oped before

IN BILE

1. Holzbach RT, Marsh M, Olszewski M, Holan K. Cholesterol solutility in bile: evidence that supersaturated bile is frequent in healthy man. J Clin Invest 1973;52: 1467- 1479.

po-

(unpublished

identification

PROMOTER

References

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bile samples but also in samples associated with cholesterol gallstone disease. Recently, in preliminary studies

CRYSTALLIZATION

SM. bile.

548

ABEI ET AL.

GASTROENTEROLOGY Vol. 104, No. 2

activity from human gallbladder bile: role in the pathogenesis gallstone disease. Hepatology 1990; 1 1:525-533.

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19. Harvey PRC, Upadhya GA, Strasberg SM. lmmunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 199 1;266: 13996- 14003. 20. Busch N, Tokumo H, Holzbach RT. A sensitive method for determination of cholesterol crystal growth using model solutions of supersaturated bile. J Lipid Res 1990;3 1: 1903- 1908. 21. Turley SD, Dietschy JM. Re-evaluation of the 3a-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978; 19:924-928. 22. 23.

24.

25.

26. 27.

Bartlett GR. Phosphorus assay by column chromatography. J Biol Chem 1959;234:466-468. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. J Clin Chem 1974;20:470-475. Castell JV, Cervera M, Marco R. A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A reexamination of the condition of reaction. Anal Biochem 1979;99:379-39 1. Yamazaki K, Powers SP, LaRusso NF. Biliary proteins: assessment of quantitative techniques and comparison in gallstone and nongallstone subjects. J Lipid Res 1988;29: 1055- 1063. Regnier FE. High-performance liquid chromatography of proteins. Methods Enzymol 1983;9 1: 137- 190. Rivier J, McClintock K. Isolation and characterization of biologically active peptides and proteins using reversed-phase HPLC. In: Kerlavage AR, ed. The use of HPLC in receptor biochemistry. New York: Wiley, 1989:77-105.

28. Ling N, Yao S-O, Ueno N, Esch F, Denoroy L, Guillemin R. Isolation and partial characterization of Mr 32,000 protein with inhibition activity from porcine follicular fluid. Proc Natl Acad Sci USA 1985;82:72 17-722 1. 29.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 30. Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced sensitivity. Anal Biochem

1981;117:307-310. 31. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide

gels to nitrocellulose

sheets: proce-

dure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4354. 32. Smith BF, LaMont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259: 12 170- 12 177. 33. York WAS, Darvill AG, McNeil M, Stevenson TT, Albersheim P. Isolation and characterization of plant cell walls and cell wall components. Methods Enzymol 1985; 118:3-40. 34. Osawa T, Tsuji T. Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem 1987;56:2 l-42. 35. Halpern Z, Dudley MA, Lynn MP, Nader JM, Breuer AC, Holzbach RT. Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase. J Lipid Res 1986;27:295-306. 36. Abei M, Kawczak P, Nuutinen H, Schwarzendrube J, Holzbach RT. Identification of the 42-kD biliary cholesterol crystallization-promoting glycoprotein as a,-acid glycoprotein (abstr). Gastroenterology 1992; 102:770A.

Received February 28, 1992. Accepted August 21, 1992. Address requests for reprints to: R. Thomas Holzbach, M.D., Gastrointestinal Research Unit, Research Institute and Department of Gastroenterology, Cleveland Clinic Foundatlon, 9500 Euclid Avenue, Cleveland, Ohio 44195-5218. Supported by Research grant DK-17562 from the National Institutes of Health. Drs. Abei and Nuutinen were supported In part by the Research Institute, Cleveland Clinic Foundation. Dr. Nuutinen was also supported in part by the Finnish Cultural Foundation and by the Paulo Foundation (Helsinki). Carbohydrate studies and analysis were supported in part by National Institutes of Health Biomedical Resource Center Program Grant l-P41-RR05351-02 to the complex carbohydrate Research Center, University of Georgia. Dr. Svanvlk’s participation was supported by the Swedish Medical Research Council. This work was presented in preliminary form at the annual meeting of the American Association for the study of Liver Diseases, Chicago, Illinois, November 5, 1991, and published as an abstract in Hepatology

1991;14:136A.

The authors thank Janeth Sperry for excellent assistance in manuscript preparation.