Identification of Lectin-like Substances Recognizing ...

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Jul 10, 1984 - 7$03.OO/O. For personal use only. on March 6, 2017. by guest ... stoned at 4 #{176}C.The average diameter of gold particles measured by.
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Identification Residues

of Lectin-like of Glycoconjugates of Marrow By Mikio

Plasma

membrane

the

to

sugar

identified bound lar

several

cell

and

sinus

is the

that

Neoglycoprotein

covalently

binding

pyrannose

form

gold

bound

then

‘C.

inhibited

DLASMA

I

with

the

the

fucose,

or

and

in galactosyl,

sugar residues systems.’ The

system

recognizing

system

may

and

selective

have

binding

the

in

internaliza-

capa-

of certain

and

the

other

mannosyl-

These

function

in the

without

nal

is the

site

and with

mannosyl colloidal

but it has between

endothelial

mem-

and the terminal be involved.8’#{176} have synthesized

containing

galactosyl,

residues. Using these gold, we have demonstrated

sugar

labeled

uptake of the demonstrated

fucosyl,

probes labeled the presence

probe

perfused

into

MATERIALS Preparation The developed Blood.

galactosyl-containing by the uptake the

AND

marrow

probe of the

l25I

recognition

of these

for the

in our Vol65,

by Grune

synthesis

laboratory,

This

method

No5(May),

1985:pp

been

in

V, Sigma

and

labeling

then

microscopic

probes,

recently in detail.”

Chemical these

to a carrier sugar

surface

of specific

circulating

provide

inter-

and

cell-

a mechanism

for

to

galactose,

of reactive

bovine

St Louis)

synthetic

probe.

is essential

L;

for

Polyscience, of

fucose

aqueous

hours

‘/2

most

which

time

of the odor

The

the

gold

was

(Polyscience)

the

Veterans

University

for This

mixture

solution

to remove stored

at

was the

free

20 #{176}C.

-

determined

by the was

ratio

38 and

to a mol/L

18 hours,

concentration

meain

the

50.

by reducing 25 mL

of

Medical of

was

Probe

Administration

Medicine,

pH

slowly

sugar-protein

Gold with

bubbled

in 0.01 reaction

was

The

prepared

added

This

Protein

to be between

solution

The

then

preparations

ofColloidal

removed.

were

‘2

of

mixture

was

chloride

neoglycoproteins

method.’3

reaction

at 9.0.

sodium

procedure.

added aqueous

Nitrogen

temperature

maintained

in these

80%

Mmol/L)

at room

was

was calculated

colloidal

of

stirring.

Lowry’s

Preparation

From

while

pH

acid

the

preparation

The

pH

synthesized

by

was 0.44

9.0,

was derivative

in

was

(BSA,

0. 1 5 mol/L

concentration

AuCI4

of the

the solution was evaporated in to adjust the volume to 5 mL.

derivative

stirring

against

oncinol-sulphunic

ment

mL),

Pa)

The

protein

to further

dialyzed

sured

of monosac-

the diffusion

mmol/L)

temperature.

ofsugar

carrier

(I5

buffer

sugar.

an electron

binding

derivatives.

at room

until

solution

of the

was subjected then

(BSA; proteins

with

The

p-aminophenyl

(0.185

isothiocyanate I

the solution

during

carrier

Warnington,

solution or

to obtain

solution

albumin

or other

to prevent

adjusted to 6.0 with 1 N HCI and vacuo. Distilled water was then added This

monosaccharide

serum

neoglycoproteins (I2I)

l0-mL

mannose,

through

to

Co.

protein

(80

stirring

ethanol

binding

molecules.

Ihiophosgen

1,000

mL

1% sodium

Center

Mississippi

of 0.01% citrate

and

as

Depart-

Medicine

Center,

Jackson. by National

institutes

ofHealth

grant

No.

AM-30142

M. T.

Medicine,

1163-1171

luminal

the

Inc.

form,

or a radioactive

chanides

Submitted

elsewhere

the

in

for

glycoproteins.

on covalent

pynannose

Fraction

Address

described

may

& Stratton,

is based

derivatives,

10

METHODS

of neoglycoprotein has

of

abdominoted

evidence

on

the con-

the was

capable

residues

for

further

into

provide

membrane

galactosyl

membrane,

uptake

substance

endothelial

specific

Supported

circulation.

of Neoglycoproteins

method

data

and

Hexose

three

of a membrane component in the marrow sinus endothelium specifically recognizing and binding galactosyl residues of the galactosyl-terminating neoglycoproteins. Neither fucosylnor mannosyl-containing neoglycoprotein probes bound to the marrow endothehum. The was further

These

BSA-gold

responsible was

specific

glycoproteins

borate

probes

highly

bound

with

or other

nature of this selectivity is not known, postulated that specific interactions

brane (lectin-like substances) residues ofglycoproteins may In the present study we

was

fucosyl-

did

to endothelial

of 125I-galactosyl-BSA but

with

small

recognition

sinuses

of

Nor

galactosyl-BSA

action

The been

components

bind moiety

of a lectin-like

1985

and

endothelium.

of

femurs.

presence

then

sugar-recognizing

uptake

Small

and

mannosyl-BSA

the

galactosyl

by perfusion

aorta.

of massive cellular and molecular traffic, and there is considerable evidence to indicate that it may regulate both the selectivity and the magnitude of this traffic.4

neoglycoprotein

The

of marrow

compo-

glycoproteins marrow

to

residues

that

was stirred

of bone

bind

galactosyl

binding.

a

in several cell the galactosyl-

and

Gold-labeled

not

unlabeled

fucosyl,

macrophages.3

a biologic

uptake

was

glycoconjugates

mannosyl,

in hepatocytes2

glycoconjugates. The endothelium

The

and

excess

have been demonstrated most notable of these are

recognizing nents

and of

by

to activated

4 ‘C

did

tibias

Galactosyl-BSA at

BSA

firmed of

mannose.

gold.

binding

molecu-

surface

synthesized (BSA)

Tavassoli

confirming

bone

COMPONENTS

of recognizing

terminating

were

presence

the

sugar-recognizing

membrane

Both in

of

the

albumin

MEMBRANE

ble

of

colloidal

endothelial

at 37

were

galactose.

labeled

to

internalized tion

of

in

membraneand

studied

presence serum

or

cellular

we

probes

bovine

implicated

and Mehdi

galactosyl-BSA.

been

endothelium

of massive

the

binding

recently

soluble

the

specific.

for

systems.

was

site

is often

endothelium

probe

of

Because

sinuses

traffic

and

uptake

Kataoka

of specific have

systems

specific

glycoproteins.

marrow

capable

of glycoproteins

in

recognition

components

residues

Substances Recognizing Galactosyl on the Plasma Membrane Sinus Endothelium

© /985

July reprint Veterans by Grune

0006-4971/85/6505-001

10,

1984;

accepted

requests

Nov

to Dr Mehdi

Administration & Stratton,

Hospital,

9, 1984. Tavassoli, Jackson,

Department MS

of 39216.

inc.

7$03.OO/O 1163

From www.bloodjournal.org by guest on March 6, 2017. For personal use only.

1164

KATAOKA

described.”’4 pH

To

required

constructed The

determine

over

optimal

range

and

protein

was determined

protein/mL

of colloidal

determined

neoglycoproteins gold,

mg/mL)

was had

been

tion6

and

moreover

Dc

Mey,’7

who

mL

two

further

minimizes

the

pH

the

possible

anionic

glycocalyx

protein

complex

6.5

glycol

of the

sugar

addition

led

to a change

work

repulsion

then

stabilized

groups

in the

of specific

30 minutes

with

0.02%

gold

and PEG.

They

particles

particles

measured

was

does

not

18.5

measuring

the

the

assured

about

number BSA

similarly

labeled

perfusion,

the

20 minutes sized)

and

to

during

then

remove

storage.

They

tion

corresponding

520

nm. this

labeling. of

milliliter.

then

The

by

(obtained

from

of gold

(obtained

.g/mL figure

was

the specific

activity

The

of neoglycoprotein

method.’8 (14.9

U/mL;

PBS

(pH

room 0.5

Five

hundred The

temperature mmol/L

ing a total

for (carrier

of I mCi.

The

as 95. g for

concentrato

done NJ)

was

Thin with

in a Zeiss

10 electron

Perfusion

of

in 1 mL 30 L

England specific

pH

mmol/L

Nuclear, activity

ofthe

7.0,

Boston) protein

times

sized

with

intrapenitoneal

injection

g body

weight)

of 6 mg/lOO

were g body

a period

addition

of

starting

buffer

was gently

taken

in a Petri

small

blocks

blocks

similarly

dish

so as to

were

cacodylate

in graded to

further

buffer

buffered

alcohol

(pH

1% 0504

and

maintain and

embedded

the

of gray-silver acetate

in

orientation

to silver lead

in The

20 mL

of buffer.

mL)

aorta

to

of 4 mL/min

for

rate to

of

five

of

range

citrate,

(10

and

were

observed

inferior

femurs

and

tibias

were

in a gamma were and

done during

counter.

for

IS minutes of

its

at was

radioactivity

five minutes

with

the

that

cannula

radioactivity

removed

and

was

counted

studies

nonradioactive

perfusion

at then

perfused

through and

The

was

volume

Inhibition

by perfusing the

legs.

Perfusion

amount

also

cannula

as described,

for another cava,

using

of the both

g/mL)

collected

vena

tip

PBS,

total

continued was

experiments

perfuse

The

the

was

in the

minutes.

2 mL/min.

Perfusate the

as

Dulbecco’s

calculate

perfusion in

before

that

with

radioactivity

experiments

except

started

order

The

in

prepared

‘251-galactosyl-BSA

perfused. placed

similarly perfusion, abdominal

flow

recorded

at

(I

probe

of the

radioreactive

between

three

for

in these mg/

probe.

30 jzL of and

100

Morphometric

Methods

containwas

For

0.6

Perfusion (1 80 to 200

the

with

37 #{176}C at the

anestheweight

electron

experimental The

and

factor. the total

luminal

length

micrograph

magnification this

condition,

available

On

coated of

each studied.

the

rats

7.3,

Neoglycoprotein

again

rate

continued that

mCi/mg.

Sprague-Dowley

in was

flow

were

Tissue

over

placed

These

mol/L

with

sections

were

placed

perfusion the

incubated H2O2,

rigidity

microscope.

/25j

animals

the

of PBS,

of 4.5

block.

molds

uranyl

neoglycoprotein-gold

lactoperoxidase three

cortex was into

in 0.1

dehydrated

stained

counted.

were

microdissected

embedding

sinuses.

lactoperoxidase

washed

using

obtained,

was per

and

beads

were

812

The to

to Horisbergen

of immobilized

pH

hour.

vascular

provide

neoglycoprotein

by the

tissue

postfixed

by

by the the

the

in situ

solution. its bony

was

then

by

at

glutaralde-

necessary

done

a

period

buffer,

estimated

was

with

fixed (2.4%

At perfu-

of

necessary particles

of

and

was

to both

marrow in each

clear

continued

cacodylate

were

The sinus

results.

desired

solution was

and

1% glutaraldehyde

Specimens Epon

and

central

tissue

of 4

presence micro-

the

to obtain

mol/L

rate

formed

reading).

was

New final

for one

the

cutter.

overnight

was

30 minutes free;

that

removed,

alter

the

complex

then

fixative

with

7.3)

and

a bone the

fixed

also

wavelength

according

lactoperoxidase

iodide,

include

by

was

not

the

mg/mL)

neoglycoprotein-gold femur

and

have

gold

the The

containing

of ‘251-neoglycoprotein)

Freehold,

washed

potassium

zL of Na’25I

10”

and

The

then

of fixative

(I

space.

for

studies

neoglycoprotein

100

studies.

was

concentration

microliters

Worthington, 7.0).

g

60 mL

flow

was

of fixation

containing

the

complex Karnovsky

Inhibition

limbs

better-quality

did

necessary

in 0.1

Usually

at

provided

Finally,

adequacy

I 5 minutes.

unlabeled

to a neoglycoprotein

x

spectrophotometric

of Neoglycoprotein

of

on

empirically,

calculated

the

lodination labeling

7.7

of leg muscles.

PBS

was

The

the lower PBS

BSA

Perfusion

modified

1% paraformaldehyde mosm).

off using

a gold

dilution

hyde, 770

(0.2-zm-pore

at the

corresponded and

measuring from

found,

a threefold

which

8.4

to obtain of I . 100

was

Usually

latter

Rossetit

diluted

absorbance

concentration,

concentration and

were

of

37 #{176}C fixative)

low-temperature

and

Dulbecco’s

was

of 2 mL/min.

of ice-cold

of the

42 #{176}C. For

and

cava.

rate

was

37 #{176}C. For

exception

0.1%

generally

vena

from

the cannulation.

ice-cooled,

necessary

the

flow

at

retrieved

U of hepanin

the circulatory

of perfusion

the inferior

the

with

from

not

of 1%

at 400

might

started or without

was

was 500

the was

perfusion.

was

blood

cannula

of the

4 #{176}C and

were

the

perfusate

this

minutes

perfusion

bath

with out

at (with

fluids

incision,

tip

before

both fluids

of neoglycoprotein-gold

indicated

filter

that

from

of

for control

a millipore

sate

particles

centrifuged

aggregates

the

large

calculated

done

TAVASSOLI

abdominal

clotting,

immediately

in ice during and

but

diameter

particles,

was

gold again

through

concentration

tissue

obtain

colloidal were

large

to the

This

optimal

filtrated any

to the

particle

five

determined

BSA

bound

least

particles I 7,500

solutions

2tllabeled

pen gold with

then

of these

protein

solutions

to gold

was

kept

in the

and

use

(EM)

vein

prevent

in a water

to wash

graphs,

in which

average

tail

solution

of Dulbecco’s

belongs

that

using

molecules

was

sizing

of BSA

perfusate

was

PEG

for

The

artery. To

gold-

removing

microscopy

coat

Experiments

the

the

perfusion

mL/min

solution

After

at 4 #{176}C. The

This

8% of the added

by

g for 20 minutes, gold saline (PBS) at

Monodispersity

of protein

Before

nm.

The

availabil-

neoglycoprotein-gold

electron

protein

label.

251

by EM.

usually

3.6 nm.

±

include

The studies

in 50 mL

stoned by

of 2 mL

also

to

the

all perfusion

this.

and The

confirmed

in 510

suspended

were

addition

was

to the

absorbance

finally

probe

surface.

or aggregation

aggregates by centrifugation at 400 were washed twice by phosphate-buffered for

the

were

and

femoral

prewarmed

of

a midline

all perfusion

0.006%

pH with

to neutral

20 mol/L).’5’6

probe

lectins

in the

is close

were

experiments,

Through cannulated

cava.

perfusion

(I

mol/L

optimal

right

experiments,

aggregaaccording

the

between

pH

of 0.2

in agreement

by the

column

the

protein,

endothelial

Carbowax;

affinity

the

in our the

this

minimizes

that

used on

addition

P1 of the are

charge

with water

solution,

pH

our data

present (PEG;

lectin-immobilized

by the This

and

to 7.0

was

polyethylene

gold

demonstrated

is 6.0 to 6.5,

Moreover,

to 7.0 to the

the

was

vena into

The

gold

label

in deionized

minutes.

is close

To

fucosyl-BSA)

of colloidal

to 6.5

for has

galactosyl-BSA

ity

galactosyl-,

nm.

in the inferior

injected

10 g of

by colloidal

520

‘25l-neoglycoprotein

adjusted

mixed

formation

the

the

than

aorta

placed

minimized

more

pentobarbital.

abdominal

were

concentrations.I5l6

6.5 and at

sodium

and

curves

that

Aggregate

to 200

concentration standard

protein

as pH above

of

added

and

and

spectrophotometry

5 mg

K2CO3

protein gold,

concentration

(mannosyl-,

colloidal which

of pH

gold.

by

optimal

of colloidal

a wide

pH

aggregation was

the

for stabilization

AND

length pits

(CPs)

surface

This

using

converted

was

number

and

of endothelium

expressed of gold

uncoated a

magnifying

pits

to the

and was

real

length

in micrometers particles (UPs) glass.

and were Only

six

animals

measured real

the

areas

the

length.

numbers

scored the

on

using

on

of the that

From www.bloodjournal.org by guest on March 6, 2017. For personal use only.

GALACTOSE

actually

RECEPTORS

formed

a depression

lntracytoplasmic lumen

length

of endothelium

ben of pits

with by the

100

zm real

CPs

and

UPs gold

statistical

analysis

To 200

real

surface nelles

percentages

as pits.

The pletely

connected

UPs

the

were

per

unit

scored

expressed

whether

as per

or not

the

About

still

in

Those

the

considered

probes with

within

lumen

the

the

were side

were

100 to

were

cytoplasmic

or within

proper

luminal

lumen

considered

This

enumer-

on the

orga-

that

hemopoietic

as abluminal.

mal

in Nonperfused

Marrow

The structure of marrow sinus endothelium in norrats has previously been reported,9”#{176}”9’2#{176} and the

observations vious findings.

was

pits and vesicles, nal sides. Most, with

the

the presence on both but not

frequency

of numerous

the all,

of CPs

higher

UPs (Table I). Some pits showed by a diaphragm. Diaphragmed noted. Binding

ofNeoglycoproteins

Galactosyl-BSA of the 1 1 .3

membrane

(n

endothelium)

probe

29)

=

with

that

per

of 1 50 zm real D).

mannosyl-BSA surface

galactosyl-BSA)

length

(P

and

Galactosyl-BSA

4

Inhibition

Mannosyl-BSA Fucosyl-BSA