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