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Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase

Authors:
  • The Institute for Molecular Medicine

Abstract

Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).
THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
0
1984
by
The
American Society
of
Biological Cbemista,
Inc.
Vol.
259,
No.
4,
Issue
of
February
25,
pp.
2283-2290
1984
Printed
in
d.S.A.
Metastatic Melanoma
Cell
Neparanase
CHARACTERIZATION
OF
HEPARAN SULFATE DEGRADATION FRAGMENTS PRODUCED
BY
B16
MELANOMA ENDOGLUCURONIDASE*
(Received for publication, August
30,
1983)
Motowo Nakajimazg, Tatsuro
Irimural7,
Nicola
Di
FerrantelJ
**,
and Garth
L.
Nicolson$
Sf:
From the $Department
of
Tumor
Biology,
The
University
of
Texas-M.D. Anderson Hospital and
Tumor Institute,
Houston, Texas 77030 and
the
)/Laboratories
of
Connective
Tksue
Research,
Department
of
Biochemistry, Baylor
College
of
Medicine, Houston,
Texas
77030
Heparan sulfate
(HS),
a
prominent component of vas-
cular endothelial basal lamina,
is
cleaved into large
M,
fragments and solubilized from subendothelial basal
lamina-like matrix by metastatic murine
B16
mela-
noma cells. We have examined the degradation prod-
ucts of HS and other purified glycosaminoglycans pro-
duced by
B16
cells. Glycosaminoglycans 3H-labeled
at
their reducing termini or metabolically labeled with
[36SJsulfate were incubated with
B16
cell extracts in
the absence or presence of D-saccharic acid 1,4-lac-
tone,
a
potent exo-&glucuronidase inhibitor, and gly-
cosaminoglycan fragments were analyzed by high
speed
gel permeation chromatography.
HS
isolated
from bovine lung, Engelbreth-Holm-Swarm sarcoma,
and subendothelial matrix were degraded into frag-
ments of characteristic
M,,
in contrast
to
hyaluronic
acid, chondroitin 6-suIfate, chondroitin 4-sulfate, der-
matan sulfate, keratan sulfate, and heparin which
were essentially undegraded. Heparin, but not other
glycosaminoglycans, inhibited
HS
degradation. The
time dependence of
HS
degradation into particular
M,
fragments indicated that
HS
was cleaved at specific
intrachain sites. Ln order to determine specific
HS
cleavage points,
HS
prereduced with NaBH4 was in-
cubated with
a
B
16
cell extract and
HS
fragments were
separated.
The
newly formed reducing termini
of
HS
fragments were then reduced with NaBISHJa,
and
the
fragments hydrolyzed to monosaccharides by trifluo-
roacetic acid treatment and nitrous acid deamination.
Since ‘H-reduced terminal monosaccharides from
HS
fragments were overwhelmingly
(>go%)
L-gulonic
acid, the HS-degrading enzyme responsible
is
an en-
doglucuronidase (heparanase)
.
During blood-borne tumor metastasis formation, malignant
cells must invade the vascular endothelial cell layer and its
underlying basal lamina
(1,2).
In
vitro
metastatic tumor cells
*
The costs of publication
of
this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked
“aduertisement”
in accordance with
18
U.S.C.
Section 1734
solely to indicate this fact.
5
Supported by American Cancer Society Institutional Grant
IN-
34.
II
Supported by American Cancer Society Institutional Grant IN-
121B.
**
Supported by Research Grant R01-AM26482 from the National
Institutes of Health, United States Public Health Service.
$3
Supported by Research Grants R01-CA28844 and R01-
CA28867
from the National Institutes of Health, United States Public
Health Service. To whom correspondence should be addressed at
Department
of
Tumor Biology-108, The University
of
Texas-M.D.
Anderson Hospital and Tumor Institute, 6723 Bertner Avenue,
Hous-
ton. Texas
77030.
easily penetrate intact blood vessel walls
(3,4)
and endothelia1
basal lamina-like matrix
(5-7)
composed of glycoproteins such
as fibronectin
(8-10)
and laminin
(11-13),
collagens, partic-
ularly type
IV
(14-16),
and sulfated proteoglycans
(17-19).
It
is thought that metastatic cells penetrate the endothelial basal
lamina using degradative enzymes specific for these basal
lamina components
(1,
2, 20-23).
Vascular endothelial cell extracellular matrix has been used
as
a substrate for tumor cell degradation studies
(6,
17-19,
24).
Highly metastatic cells solubilize the major components
of endothelia1 basal lamina such as fibronectin, laminin, and
HS’
(6,
17-19,24).
We have found that the average
M,
of the
HS
released from endothelial basal lamina-like matrix by
B16
melanoma cells is approximately one-third the
M,
of the
original molecules in the untreated matrix, suggesting that
B16
cells possess an endoglycosidase capable of degrading
HS
into intermediate
M,
fragments
(17).
Highly invasive and
metastatic
B16
sublines degrade sulfated glycosaminoglycans
(S-GAGS)
of
the basal lamina-like
matrix
at higher rates than
B16
cells of lower metastatic potential
(6,
18).
B16
melanoma cells also fragment purified
HS
(18,
19).
Intact
B16
cells or
B16
cell extracts from sublines
of
high
lung colonization potential degrade purified
HS
at higher
rates than
B16
cells of poor lung colonization potential
(18).
Here we demonstrate that these activities are due to a
HS-
specific endoglucuronidase in
€316
melanoma cells
by
char-
acterization of
HS
degradation products using gel chromatog-
raphy
(25-27)
and high speed gel permeation chromatography
(28)
and by determining the reducing terminal saccharides
of
IiS
degradation fragments.
EXPERIMENTAL
PROCEDURES
Materials
CeMs and Cell
Culture-Highly invasive and lung metastatic murine
B16 melanoma subline
(B16-BL6)
was obtained from Dr.
I.
J.
Fidler
(The University
of
Texas-M.D. Anderson Hospital and Tumor Insti-
tute, Houston,
TX).
Melanoma cells were grown on plastic tissue
culture dishes in a
1:l
mixture of DME/FlP (Gibco, Grand Island,
NY),
supplemented with
5%
heat-inactivated fetal bovine serum
(Reheis, Kankakee,
IL),
under humidified conditions with 95% air-
5%
CO,.
BAE
cells, obtained from Dr.
D.
Gospodarowicz (University
of
California, Medical Center, San Francisco, CA), were cultured in
The abbreviations used are: HS, heparan sulfate; GAG($, glycos-
aminoglycan(s); C4S, chondroitin 4-sulfate;
C6S,
chondroitin 6-SUI-
fate; DS, dermatan sulfate; HA, hyaluronic acid;
KS,
keratan sulfate;
SAL, D-saccharic acid l,4-lactone; BAE cells, bovine aortic endothe-
lial cells;
BCE
cells, bovine corneal endothelial cells; DME, Dulbecco’s
modified minimum essential medium;
F12,
Ham’s
F-12
medium
DPBS,
Dulbecco’s phosphate-buffered saline; EHS, Engelbreth-
Holm-Swarm;
PYS,
parietal
yolk
sac.
2283
by guest, on July 13, 2011www.jbc.orgDownloaded from
2284
Metastatic
Tumor
Cell Heparanase
DME/F12 medium supplemented with 10% heat-inactivated fetal
bovine serum (Biocell, Carson, CA), 500 ng/ml of fibroblast growth
factor (29), and 0.1 mM nonessential amino acids. The mouse EHS
tumor was obtained from Dr. L.
A.
Liotta (National Cancer Institute,
Bethesda, MD) and maintained in C57BL/6 mice by subcutaneous
implantation of minced tumor tissue. After 4 weeks of growth tumors
(7-8 g) were excised and dissected. The tumor tissues were then
washed with DPBS (Gibco) and cultured in DME/F12 medium sup-
plemented with 10% heat-inactivated fetal bovine serum (Biocell)
and 25 pg/mI of gentamicin (Elkins-Sinn, Cherry Hill, NJ).
Glycans-Bovine lung HS was purified according to Cifonelli and
Dorfman and Schiller et al. (30, 31), and its average
M,
(-34,000)
was determined by sedimentation equilibrium. Specimens of HA from
human umbilical cord, C4S from rock sturgeon notochord, C6S from
human umbilical cord,
DS,
and heparin from porcine mucosal tissue
were prepared under a United States Public Health Service National
Heart Institute grant and kindly donated by Drs.
M.
B. Mathews,
J.
A.
Cifonelli, and L. Rodh (University of Chicago, IL). Heparin from
porcine intestinal mucosa and bovine lung, and
KS
from bovine
cornea were obtained from Sigma. C6S from shark cartilage and HA
from human umbilical cord were obtained from Miles Laboratories
(Naperville, IL). Some of these GAGS were submitted to further
purification by gel chromatography
on
columns of Sephadex G-75,
Sephacryl S-200, or S-300 (Pharmacia). Monosialosyl biantennary
complex-type glycopeptide UB-I-b was prepared from thyroglobulin
(Sigma) according to Yamamoto
et
al. (32). Tri-N-acetylchitotriose
was prepared from crab shell chitin according to Rupley (33).
D-Galactose, D-XylOSe, and 2-deoxy-~-glucose were purchased from
Calbiochem-Behring. N-Acetyl-D-glucosamine, D-glucosamine hydro-
chloride, SAL, and D-glucuronic acid lactone were purchased from
Sigma. D-Glucuronic acid was purchased from K and
K
Chemicals
(Plainview,
NY).
L-Iduronic acid was isolated from heparin.
Methods
Cell Extracts-Subconfluent
B16
melanoma cells of less than eight
passages from an original frozen stock were harvested by treatment
for 10 min with 2 mM EDTA in Ca*+,Me-free DPBS. After suspen-
sion into single cells, they were washed twice by brief centrifugation
in 0.14
M
NaC1,
10
mM Tris-HC1 buffer, pH 7.5, and checked for
viability (usually >95%) by trypan blue dye exclusion. Cells were
suspended in chilled 50 mM Tris-HC1 buffer, pH 7.5, containing
0.2%
Triton X-100 at a concentration of
6
X
lo6
cells/ml. Cell suspensions
(1
ml) were sonicated for 20
s
at 4 "C at constant power using a cell
disruptor model W200R (Ultrasonic, Inc., Plainview, NY) equipped
with a microtip. The supernatant (approximately
2
mg of protein/
ml) was collected after centrifugation at 9800
X
g for 5 min. Protein
contents in the centrifuged extracts were determined by a modifica-
tion of the Lowry technique (34) to correct for the presence of Triton
X-100 in the samples.
Glycosaminoglycan Degradation-Purified GAG was mixed with
the centrifuged melanoma cell extract in
0.1
M
sodium phosphate
buffer (pH
6.0),
0.15
M
NaC1, 0.2% Triton X-100, 0.05% NaN3
(reaction buffer A). The incubation was carried out at 37 "C with
occasional gentle mixing. The incubation was terminated by chilling
the solution to 4 "C and adding of
50%
trichloroacetic acid
to
a final
concentration of 5%. After centrifugation at 9800
X
g for
5
min, the
supernatant was neutralized with
1.0
N
NaOH and submitted to
further analysis (see below).
Gel
Chromatography-HS was incubated with B16 cell extracts or
heat-inactivated B16 cell extracts in reaction buffer
A
(pH
6.0),
and
reactions were terminated as described above. After removal of tri-
chloroacetic acid-insoluble materials, the supernatants were imme-
diately neutralized and passed through small columns of AG 50W-
X8 (H+ form). Acidic fractions were collected on ice, neutralized with
1.0
N
pyridine, and lyophilized. The lyophilized samples were dis-
solved in
1
ml of 0.2
M
pyridine-acetate buffer, pH 5.0, and applied
to a Sephacryl S-200 column (0.9
X
107 cm) previously equilibrated
with the same buffer. Elution was performed
at
a rate of
10
ml/h at
4
"C;
effluent fractions
(1
ml) were collected and analyzed for uronic
acid by the method of Bitter and Muir (35).
Preparation
of
3H-labeled
GAGS-'H-Labeling of GAGS was per-
formed as follows. One milligram of purified GAG was reduced with
2 mCi of NaB[3H]4 (340 mCi/mmol; New England Nuclear) in
0.1
M
sodium borate buffer, pH
8.0,
at 25 "C for 5 h. After acidification to
pH 5 with acetic acid, the mixture was chromatographed on a column
(0.9
X
105 cm) of Sephacryl S-200 or S-300 equilibrated with 0.2
M
pyridine-acetate buffer, pH 5.0. Individual 3H-labeled GAGS of spe-
cific M, were collected and lyophilized. After dissolving in water, 3H-
labeled GAG was precipitated by the addition of ethanol and NaCl
to
a final concentration of
80%
and 10 mM, respectively. Precipitated
GAG was then washed with
80%
ethanol and lyophilized to completely
remove pyridine. These steps yielded 3H-labeled GAGS with specific
radioactivities of 500
to
1100
cpm/pg of GAG.
Isohtion of 36S-labeled
HS
from
EHS
Sarcoma and BAE Suben-
dothelial Matrix-Primary cultures of minced EHS sarcoma tissues
(approximately
3
g) were labeled for
48
h with 25 pCi/ml of Na2[36S]
0,
(New England Nuclear) in sulfate-depleted DME medium con-
taining
10%
heat-inactivated fetal bovine serum in 10-cm tissue
culture dishes. The following steps were carried out at 4 "C except for
pronase and chondroitinase ABC digestion. "S-labeled tissues washed
with DPBS were frozen and thawed twice and sonicated in 50 mM
Tris-HC1 buffer, pH 7.5.
A
10 times volume of acetone was added and
extraction allowed to proceed for
3
h with mixing.
At
the end of the
extraction, insoluble materials were collected by centrifugation, and
the sample was re-extracted with acetone and dried completely. The
residue was suspended in 20 ml of
1
mg/ml of Pronase (Calbiochem-
Behring) in 0.15
M
NaC1, 7 mM CaCI2, 0.05% NaN3,
10
mM Tris-HC1
buffer, pH 7.5, for 40 h at 37 "C. The mixture was then chilled in an
ice bath and mixed with one-fifth volume of 50% trichloroacetic acid.
After removal of trichloroacetic acid-insoluble materials by centrifu-
gation for 20 min at 9800
X
g,
the supernatant was neutralized with
0.1
N
NaOH and dialyzed against water. The dialyzed solution was
applied to a small AG 50W-X8 (H+ form) column and eluted with
water. Acidic fractions were collected, neutralized, and lyophilized.
For chondroitinase ABC treatment, lyophilized materials were
dis-
solved in 4 ml of water and then mixed with
1
ml of chondroitinase
ABC (Miles,
5
units/ml) in 0.25
M
Tris-HC1, 0.3
M
sodium acetate,
0.25
M
NaCI, and 0.05% bovine serum albumin (Sigma), pH
8.0.
After
incubation for
18
h at 37 "C, the reaction mixture was dialyzed against
water and subjected to AG 5OW-X8 cation exchange chromatography
as described above. Acidic radioactive materials were further frac-
tionated by Sephacryl S-200 chromatography. The major high M,
radioactive material was collected and lyophilized. 36S-labeled HS
was identified by agarose gel electrophoresis in 1,3-diaminopropane
acetate buffer, pH 9.0, according to the method of Dietrich and
Dietrich (36). Radioactive materials and standard GAG molecules
were detected by toluidine blue staining and autoradiography with
Kodak X-Omat AR-5 x-ray film. Purified 35S-labeled HS was resistant
to chondroitinase ABC, as well as chondroitinase AC, but was sus-
ceptible to nitrous acid deamination (37).
%-labeled HS was also prepared from the extracellular matrix of
cultured BAE cells. Confluent BAE cell monolayer cultures in IO-cm
tissue culture dishes were labeled for 48 h with 25 pCi/ml of Na2[36S]
O4
in sulfate-depleted DME medium containing 10% heat-inactivated
fetal bovine serum, 500 ng/ml of fibroblast growth factor, and
0.1
mM
nonessential amino acids. Subendothelial matrix was isolated as
described previously (9) and was then digested with
2
ml of Pronase
solution as described above. Further steps were performed as de-
scribed in the method for isolation of HS from EHS sarcoma. The
specific radioactivities of "S-labeled HS from EHS sarcoma and BAE
subendothelial matrix were
700
and 1500 cpm/pg of hexuronic acid,
respectively. Hexuronic acid content was determined by the carba-
zole-borate method (35) using D-glucuronic acid lactone as the stand-
ard.
High Speed Gel Permeation Chromatography-High speed gel per-
meation chromatography was carried out using a high pressure liquid
chromatograph system equipped with two sequential columns (0.7
X
75 cm) of Fractogel (Toyopearl)
TSK
HW-55(S) (MCB, Gibbstown,
NJ)
as described previously (28).
3H-
or =S-labeled GAG was incubated at 37 "C with a B16-BL6
cell extract in
100
pl of reaction buffer
A
(pH
6.0).
The reaction was
terminated as described before, and
100
pl of sample solution were
delivered into the injection port. Chromatographic elution was per-
formed with 0.2
M
NaCl at a flow rate of
1.0
ml/min at
55
"c
(28).
Effluents were collected each 30
s
of elution (0.5-ml volumes), mixed
with 3.4 ml
of
Hydrofluor (National Diagnostics, Somerville, NJ),
and counted
on
a Beckman 7500 liquid scintillation counter (Beck-
man Instruments, Irvine, CA).
Preparation
of
Labeled Monosaccharide Alcohol Standards-Mono-
saccharide
(1
mg) was reduced with
1
mCi of NaB[3H]4 (170 mCi/
mmol) in 300
pl
of
0.01
M
NaOH at 25 "C for 3 h, NaBH. (5 mg) was
then added, and the reaction was continued for another 2 h at 25 "C.
The reaction mixture was neutralized with
2
M
acetic acid and then
applied to a small column of AG 5OW-X8 (H+ form) and eluted with
by guest, on July 13, 2011www.jbc.orgDownloaded from
Metastatic
Tumor
Cell
Heparanase
2285
water, except for 3H-labeled D-ghlcosaminito~, which was eluted from
the AG 50W-X8 column with
1
M
ammonia. Boric acid was removed
by repeated evaporation with methanol, and radioactive contami-
nants were removed using descending paper chromatography (What-
man No.
1)
in I-butanol-pyridine-water (6:4:3) (38).
Analysis
of
Reducing Terminal Saccharides
of
HS
Degradation
Fragments-Purified HS
(5
mg) from bovine lung was reduced in 2
ml of 0.5
M
borohydride, 0.1
M
borate buffer, pH
8.0,
at 22 "C for 3 h.
Excess borohydride was destroyed by acidification to pH 5.0 with 2
M
acetic acid; the mixture was applied to a Sephacryl S-200 column
(0.9
X
107 cm) equilibrated with 0.2
M
pyridine-acetate buffer, pH
5.0, and it was then eluted with the same buffer. Eluent was monitored
by measuring hexuronic acid according to Bitter and Muir (35).
Reduced HS fractions were collected, lyophilized, and then washed
with ethanol to remove pyridine. Reduced HS (2 mg) was incubated
at 37 "C for 12 h with a B16-BL6 cell extract
(1
mg of protein) in 2
ml of reaction buffer
A
(pH
6.0)
in the presence of 20 mM D-saccharic
acid 1,4-lactone, a lysosomal @-glucuronidase inhibitor (39). The
reaction was terminated by chilling to 4
"C,
and 220
p1
of 50%
trichloroacetic acid were added. After the samples were centrifuged
at 9800
X
g for
5
min at 4
"C,
the supernatants were neutralized with
1
N
NaOH, and ethanol and barium acetate were added to final
concentrations of 80 and 0.376, respectively. The mixtures were left
at 4
"C
for 20 h, and the precipitates were collected by centrifugation
as before. Precipitates were dissolved in water and applied to a small
column of AG 50W-X8 (H+ form) eluting with water. The pass-
through fractions were lyophilized and chromatographed on Sephac-
ryl S-200 as described above.
Reducing terminal saccharide residues of HS degradation products
were analyzed as follows. Fractionated HS fragments were reduced
with NaB[3H]r as described in the method for preparation of 'H-
labeled monosaccharide alcohols. The reaction mixtures were neu-
tralized with acetic acid and chromatographed
on
Bio-Gel P-10 col-
umns
(0.6
X
20 cm) with water to isolate 3H-labeled products. Deg-
radation of 3H-reduced HS fragments to monosaccharides was per-
formed by one of the following methods:
1)
acid hydrolysis in 4
N
HCI at 100 "C for
8
h
2) acid hydrolysis in 2
M
trifluoroacetic acid,
deamination in 3.9
M
NaN02-0.28
M
acetic acid, and further acid
hydrolysis in
2
M
trifluoroacetic acid according to the method of
H66k et
al.
(40). After repeated evaporation of hydrolyzed samples in
the presence of water, the hydrolysates were dissolved in
2
ml of 0.5
M
Tris-HC1 buffer, pH 8.0, and were left at 25 "C for 24 h to convert
aldonic acid lactones into free acids (40). These samples were chro-
matographed on Bio-Gel P-2 columns
(0.6
X
90 cm) with water.
Monosacchaide fractions were collected and applied to DEAE-Sepha-
cel columns (0.5
X
30 cm). The columns were successively eluted with
50 ml of water and 50 ml of 0.4
M
pyridine-acetate buffer, pH 3.0.
Pass-through fractions were repeatedly subjected to DEAE-Sephacel
chromatography after treatment with 0.25
M
ammonia to recover
quantitatively hexonic acids in the acidic fractions. Nonacidic mono-
saccharides were analyzed by descending paper chromatography
(Whatman No.
1)
in
1-butanol/pyridine/water
(6:4:3) (38) and by
high voltage paper electrophoresis (Savant Instruments, Inc., Hicks-
ville, NY) in
0.06
M
borate buffer, pH 8.9 (41). Acidic monosaccha-
rides (aldonic acids) were identified by paper chromatography of their
corresponding aldono-1,4-lactones (42) in t-amyl alcohol/isopropyl
alcohol/water (4:1:2) (43). 3H-labeled monosaccharides were detected
by radioactivity measurement, and reduced and nonreduced mono-
saccharides were detected by silver nitrate staining after short-term
periodate oxidation according to the method of Yamada et
a/.
(44).
RESULTS
Gel Chromatographic Analysis
of
HS
and Its Fragments
Produced
by
B16
Cell Extracts-To
determine the relative
Elution profiles
of
purified bovine lung
HS
showed a sharp
single peak (Fig.
1A).
In the presence of
B16
cell extracts the
original
HS
peak decreased in amount and peaks of lower
M,
appeared. After
6
h
of
incubation, degradation components
of
M,
approximately
22,000, 12,000,
and
4,000
appeared, and
free hexuronic acids were detected (Fig.
IA).
When the incu-
bation was performed in the presence
of
SAL
(20
mM), the
E
0.3
m
In
cd
I1
A
e
i
20
30
40
50 60
70
80
Elution
Volume
(
ml
)
FIG.
1.
Sephacryl
5-200
gel chromatography
of
HS
incu-
bated with
B16-BL6
melanoma cell extract
in
the absence
or
by guest, on July 13, 2011www.jbc.orgDownloaded from
2206
Metastatic Tumor Cell Heparanase
intermediate
M,
components remained, while the appearance
of low
M,
components decreased (Fig. 1B). At the monosac-
charide-eluting position, SAL was eluted quantitatively as
indicated by carbazole-borate reaction. When the materials
in the monosaccharide fractions were analyzed by paper chro-
matography
(38,
43)
and high voltage paper electrophoresis
(41),
neither glucuronic acid nor N-acetylglucosamine was
detected. Therefore, the degradation of HS to intermediate
M,
fragments was not significantly affected by the exoglycos-
idase inhibitor, indicating that HS degradation was mainly
due to endoglycosidase(s). Higher concentrations of
SAL (40
mM) markedly inhibited HS degradation, suggesting that
HS-
degrading endoglycosidases (heparanase) may be sensitive to
high SAL concentrations.
Analysis
of
Cleavage
Products
from
GAGS
Labeled
with
3H
at
Their
Reducing
Ends-HS with 3H-labeled reducing ter-
minal saccharides were incubated with
B16-BL6
celI extracts
in the presence of
SAL,
and the
HS
degradation products
were analyzed by high speed gel permeation chromatography
(28).
In this experiment, only fragments having original re-
ducing terminal residues could be identified. High speed gel
permeation chromatography of HS degradation products and
standard glycans are shown in Fig.
2.
After
1
h
of
incubation
with a
B16-BL6
cell extract in the presence of SAL, the
amount of glycan in the original HS peak decreased, while
fragments of
M,
=
-22,000, -15,000,
and
-10,000,
appeared.
m
100
0
10
1
20
30
40
Retention tlrne
(
rnln
)
FIG.
2.
High speed gel permeation chromatography
of
HS
and
its
fragments produced by
B16
heparanase.
A,
logarithmic
plot of
M,
uersus
the retention times of standard glycans separated
on two sequential columns of Fractogel-TSK HW-556). Standard
glycans are:
a,
HA from human umbilical cord
(M,
-
230,000);
b,
C6S
from shark cartilage
(M,
-
60,000);
c,
HS from bovine lung
(M,
-
34,000);
d,
DS
from porcine mucosal tissue
(Mr
-
27,000);
e,
C4S
from notochord of rock sturgeon
(M,
-
12,000);
f,
heparin from
porcine mucosal tissue
(M,
-
11,000);
g,
monosialosyl biantennary
complex-type glycopeptide from porcine thyroglobulin
(Mr
-
2,190);
h,
tri-N-acetylchitotoriose
(M,
-
627);
i,
N-acetyh-ghcosamine
(M,
-
221).
B,
elution profiles of 3H-labeled HS
(0)
and its degradation
fragments produced by
B16
heparanase
(0).
3H-labeled HS
(5
rg,
2,500
cpm) was incubated at
37
"C
with a
B16-BL6
cell extract
(40
pg of protein) in reaction buffer
A
(pH
6.0)
in the presence of
20
mM
SAL.
3H-labeled HS and its degradation fragments were fractionated
by high speed gel permeation chromatography.
Arrows
a-i
indicate
the eluting positions of the standard glycans.
At
3
h the peaks of M,
=
-15,000,
-10,000, and
-5,400
were
demonstrable, and a fragment of M,
-
5,400
accumulated
after
6
h of incubation. These profiles are much simpler than
the gel chromatographic profiles based on total hexuronic acid
contents and strongly suggest that
B16
heparanase cleaves
bovine lung
HS
at a minimum of five intrachain sites. We
could not detect any preferences in the cleavage of heparan
sulfate at any of these intrachain sites.
The following other GAGs with 3H-labeled reducing ter-
minal saccharides were also examined HA from human um-
bilical cord (M,
-
230,000), C4S
from rock sturgeon notochord
(Mr
-
12,000),
C6S
from shark cartilage
(MI
-
SO,OOO),
DS
from porcine mucosal tissue
(Mr
-
27,000),
KS
from bovine
cornea
(M,
-
14,000),
heparin from bovine lung
(M,
-
15,000),
and heparin from porcine intestinal mucosa
(MI
-
11,000).
Incubation with a B16-BL6 cell extract was carried out under
the same conditions as the HS degradation assay. Elution
profiles of these
GAGs
(except bovine lung heparin) and some
of their degradation products are shown in Fig.
3.
Per cent of
degradation of these GAGs calculated from the decrease in
20
30
40
.*
EHS-HS
.*
*.
..
A.
A'
:'.
.
""
20
30
40
HEPARIN
,
..
..
*.
.*
.
**,
..
'*.
c*
"
L
-
20
30
40
BAE-HS
.**
.*
..
*.
"* *-
a'.
20
30
40
RETENTION TIME
(
MIN
)
FIG.
3.
High speed gel permeation chromatographic analy-
sis
of
GAGs
incubated with
B16
cell extracts.
3H- or 35S-labeled
GAG
(10
pg) was incubated with a
B16-BL6
cell extract
(60
pg of
protein) in reaction buffer
A
(pH
6.0)
for
6
h at
37
"C
in the absence
or presence of
20
mM
SAL.
Incubation products were analyzed by
high speed gel permeation chromatography.
HA,
3H-labeled
HA
from
human umbilical cord
DS,
3H-Iabeled
DS
from porcine mucosal
tissue;
KS,
3H-labeled
KS
from bovine cornea;
C6S,
3H-labeled
C6S
from shark cartilage;
C4S,
3H-labeled
C4S
from rock sturgeon;
Hep-
arin,
3H-labeled heparin from porcine intestinal mucosa;
EHS-HS,
[%]O~labeled HS from
EHS
sarcoma;
BAE-HS,
[36S]04-labeled HS
from
BAE
subendothelial matrix.
A,
GAG
incubated with a heat-
inactivated
B16-BL6
cell extract;
B,
GAG incubated with a
B16-BL6
cell extract in the presence of
20
mM
SAL;
C,
GAG
incubated with a
B16-BL6
cell extract without
SAL.
by guest, on July 13, 2011www.jbc.orgDownloaded from
Metastatic
Tumor
Cell
Heparanase
2287
TABLE I
High speed gel permeation chromatographic study
of
GAG
degradation
by
a
B16
melanoma cell extract
GAG
degrada-
t.inn'
Inhibition
of
GAG
HS
(bovine lung,
M,
-34,000)'
HS
(EHS
sarcoma,
M,
-
70,000)d
HS
(BAE
subendoth-
elial matrix,
M,
-
24,000)d
Heparin (bovine lung,
M,
-
15,000)'
Heparin (porcine in-
testinal mucosa,
M,
-
11,000)'
HA
(human umbilical
cord,
M,
-
230,000)'
C4S
(rock sturgeon
notochord,
M,
-
12,000)'
C6S
(shark cartilage,
M,
-
60,000)'
DS
(porcine mucosal
tissue,
M,
-
27,000)'
KS
(bovine cornea,
M,
-
14,000)'
""_
HS
degrada
OmM
20mM
tion*
SAL
SAL
%
95.2
88.5 21.3
92.3
90.5
NT"
96.5 93.5
NT
15.6 12.3
83.6
12.0
7.1 85.3
<5.0 C5.0 <5.0
<5.0 <5.0 <5.0
18.5 C5.0
<5.0
<5.0 <5.0
<5.0
C5.0 <5.0 <5.0
a3H-
or 35S-labeled
GAG
(10
pg)
was incubated with a
B16-BL6
cell extract
(60
pg
of protein) in reaction buffer
A
(pH
6.0)
for
6
h at
37
"C
in the absence or presence of
20
mM
SAL
and was then subjected
to
high speed gel permeation chromatography. Per cent of degradation
was calculated from the decrease in area of the high
M,
half of the
GAG
peak. The data shown are the average of triplicate samples
(S.D.
<
5.0%).
'
Ten micrograms of unlabeled
GAG
was added to the incubation
mixture
of
3H-labeled
HS
from bovine lung and a
B16-BL6
cell
extract. Per cent of inhibition was calculated from the decrease in
area of the high
M,
half of the HS peak.
e
Purified
GAGs
were labeled with NaBt3HJ4.
HS metabolically labeled with
[35S]04
was isolated from briefly
cultured tissue or extracellular matrix.
e
NT, not tested.
area of the high M, half of the
GAG
peak is listed in Table I.
In the presence of
SAL,
none of the
GAGs
was detectably
affected after prolonged incubation. For example, the elution
profile of porcine intestinal mucosa 3H-labeled heparin incu-
bated with a
B16-BL6
cell extract in the presence of
SAL
showed no alterations in
M,
from that of the [3H]heparin
control (Fig.
3).
In the absence of
SAL
heparin was only
partially degraded. Partial degradation of
C6S
was also ob-
served in the absence of
SAL
(Table I), indicating that
B16
melanoma exoglycosidases may be involved. The partial deg-
radation
of
GAGs
in the absence of
SAL
may be due to
GAG
molecular heterogeneity.
Degradation
of
P5S]04-lnbeled
HS
isolated
from
EHS
sar-
coma
or
BAE
Subendothelial Matrix-To examine the action
of
B16
heparanase on
HS
prepared from various tissues, we
have used
PYS
carcinoma
(28),
EHS
sarcoma, and
BAE
and
BCE
subendothelial matrix.*
EHS
sarcoma synthesized very
large
M,
[35SJ0,-labeled HS (average
M,
-
70,000)
during
short-term culture (Fig.
3).
HS
from
BAE
subendothelial
matrix was
M,
-
24,000;
however, cell-associated HS from
BAE
cells was much smaller (average M,
-
8000).
[36S]04-
'2.
Wang,
T.
Irimura,
M.
Nakajima,
P.
N.
Belloni, and
G.
L.
Nicolson,
Eur.
J.
Biochern.,
submitted for publication.
labeled
HS
isolated from
EHS
sarcoma was degraded at high
rates during an incubation with a
B16-BL6
cell extract in the
presence
of
SAL
(Fig.
3
and Table I), and characteristic
M,
degradation peaks appeared (average
M,
=
-24,000, -14,000,
-9000,
and
-5600)
indicating that EHS sarcoma
HS
was
discontinuously fragmented by
B16
heparanase. [35S]0d-la-
beled HS from
BAE
subendothelial matrix was also degraded
at high rates (Fig.
3
and Table
I);
however, this HS appeared
to be fragmented into larger
M,
molecules (average M,
-
8000)
than the degradation products of the other HS mole-
cules. In the absence of
SAL,
HS
fragments were further
degraded and broad fractionation peaks appeared, suggesting
the action of exoglycosidases.
Effects
of
GAGS
on
HS
Degradation by
B16
Heparame-
The interactions of
B16
heparanase with various
GAGs
were
examined using
a
heparanase inhibition assay.
GAG
was
added to a
B16-BL6
cell extract incubation mixture contain-
ing an equal amount (dry weight) of 3H-labeled HS, and the
effects of
GAGs
on HS fragmentation were determined by
high speed gel permeation chromatography. When equal
amounts of HS and 3H-labeled
HS
were present, the rate of
3H-labeled HS degradation decreased slightly below that of
3H-labeled
HS
alone (Fig.
4
and Table
I).
In contrast, the
addition of porcine intestinal mucosa heparin almost com-
pletely inhibited the appearance of intermediate
M,
HS
frag-
ments (Fig.
4).
Bovine
lung
heparin also showed this inhibi-
tory effect (Table
I).
These results indicated that
B16
hepar-
anase can bind but not cleave the major
GAG
components of
heparin. Other
GAGs (HA, C6S, C4S,
DS,
and
KS)
tested did
I
I
I
I
I
I1
ID
."...
2**..-
.
20
30
LO
Retention
time
(
min
)
FIG.
4.
Effect
of
heparin
on
HS
degradation
by
B10
hepar-
anase.
Ten micrograms of 3H-labeled
HS
from bovine lung was
incubated with a
B16-BL6
cell extract
(60
pg
of
protein) in reaction
buffer
A
(pH
6.0)
containing
20
mM
SAL
for
6
h at
37
'C
in
the
absence or presence of heparin from porcine intestinal mucosa.
After
termination of the incubation, samples were analyzed
by
high speed
gel permeation chromatography. Elution profiles are:
A,
t3H]HS
incubated with a heat-inactivated cell extract;
B,
[3H]HS
incubated
with a cell extract in the presence of
10
pg of heparin;
C,
[3H]HS
incubated with
a
cell extract in the presence of an additional
10
pg
of
unlabeled
HS;
D,
[3H]HS
incubated with a
B16-BL6
cell extract.
Arrows
a,
b,
c,
f,
g,
and
h
indicate the elution positions of the standard
glycans indicated in Fig. 2.
by guest, on July 13, 2011www.jbc.orgDownloaded from
2288
Metastatic Tumor Cell Heparanase
not produce significant inhibitory effects on B16 heparanase
degradation of HS (Table
I).
Identification
of
HS
Linkage Groups Susceptible to
B16
Cell
Heparunase-The HS linkage groups cleaved by B16 hepar-
anase were identified by analysis of reducing terminal saccha-
rides of liberated [3H]borohydride-reduced
HS
fragments. To
distinguish the newly exposed reducing termini from the
original reducing termini of intact HS, the latter were previ-
ously reduced with borohydride under mild conditions to
prevent alkaline hydrolysis. After Sephacryl S-200 gel chro-
matography of digest (Fig. 5), the fragments
of
high
M,
(fractions 25 to 38
or
DP-1) and low
M,
(fractions 39 to 52 or
DP-2) were collected separately and reduced with NaB[3H],.
These HS fragments were cleaved into monosaccharides by a
combination of mild acid hydrolysis and deamination, and the
monosaccharides were fractionated by Bio-Gel
P-2
gel chro-
matography. Approximately 90% of the tritium was recovered
in the [3H]monosaccharide alcohol fractions (Fig. 6). These
monosaccharide alcohols were separated into the acidic and
nonacidic fractions by DEAE-Sephacel anion exchange chro-
matography after converting [3H]aldonic acid lactones to the
corresponding free acids. Acidic sugar alcohols were eluted
with 0.4
M
pyridine-acetate buffer, pH 3.0, and the acidic
monosaccharide 3H activities obtained from fractions DP-1
and DP-2 were 91.7 and 94.9%, respectively. These 3H-labeled
acidic monosaccharides were identified as L-gulonic acid by
descending paper chromatography of their corresponding al-
dono-1,4-lactones in t-amyl alcohol/isopropyl alcohol/water
(4:1:2) (Fig. 7). L-Gulonic acid was produced as the reduction
product of D-glucuronic acid, but 3H-labeled L-idonic acid, the
reduction product of L-iduronic acid, was not detected. 3H-
labeled nonacidic monosaccharides were identified by de-
scending paper chromatography in
l-butanol/pyridine/water
(6:4:3) (Fig. 7) and by high voltage paper electrophoresis in
0.06
M
borate buffer, pH 8.9. By these techniques, some
of
the 3H-labeled monosaccharides were identified as 2-deoxy-
D-glUCOSe, which was obtained from D-glucosaminitol by ni-
trous acid deamination. Nitrous acid deamination of oligosac-
015L
--
DP-1
DP-2
20
30
40
50
60
Elution
Volume
(
mi
)
FIG.
5.
Sephacryl
S-200
gel chromatography of prereduced
HS
and its degradation fragments produced by
B16
hepar-
anase.
Prereduced HS was incubated with a
B16-BL6
cell extract
for 12 h at
37
"C
in reaction buffer
A
(pH
6.0)
in the presence of
20
mM SAL. The reduced HS
(A)
and its fragments
(A)
were applied to
a Sephacryl
S-200
column. Gel chromatography was performed as
described under "Methods." High
M,
(fractions
25-38
or DP-1) and
low
M,
(fractions
39-52
or DP-2) degradation fragments were col-
lected separately and submitted to further analysis.
HS Degradation Products
Sephacryl
5-200
I
1
Fraction DP-1 Fraction DP-2
Reduction with NaBC3H]4
TFA Hydrolysis
Deamination
TFA Hydro1 ys
i
s
Bio-Gel P-2
13H] monosaccharides [3H] monosaccharides
(89.1%
of
total [3H])
(90.6%
of
total [3H])
A
Non-acidic Acidic Non-acidic
A
Acidic
DEAE-Sephacel
2-deoxy-0- L-gulonic 2-deoxy-0- L-gulonic
glucose acid glucose acid
Unknown Unknown (4.5%)* (91 .7b)* (3.4?3*
(94.9%)*
(3.8%)* (1.7%)*
FIG.
6.
Identification of the reducing terminal saccharides
of
HS
fragments produced by
B16
heparanase.
*,
total [3H]
radioactivity recovered as [3H]monosaccharides was taken as
100%.
TFA,
trifluoroacetic acid.
0 5
10
15
20
0
~~ ~
IO
20
30
Migration DistmcC
(em)
Migration
DIsUnce
(em)
FIG.
7.
Paper chromatography of the reducing terminal
saccharides of
HS
fragments liberated by
B16
heparanase.
Left,
paper chromatography of acidic monosaccharides in t-amyl
alcohol/isopropyl alcohol/water (4:1:2).
A,
standard saccharides:
I,
L-
gulonic acid;
II,
L-idonic acid;
III,
L-gulonolactone;
IV,
L-idonolac-
tone.
B,
3H-reduced acidic monosaccharides obtained from the reduc-
ing terminal saccharides of low
M,
fragments (DP-2 in Fig.
5)
by
DEAE-Sephacel chromatography.
C,
the same acidic monosaccha-
rides after lactonizing
(43).
Right,
paper chromatography of nonacidic
monosaccharides in
1-butanol/pyridine/water
(64:3).
D,
standard
saccharides are:
V,
D-ghCOSaminitO~
VI,
2-deoxy-D-glucitol;
VII,
2-
deoxy-D-glucose.
E,
deamination product of 3H-reduced D-glucosa-
mine.
F,
3H-labeled neutral monosaccharides obtained from low
M,
fragments (DP-2 in Fig.
5).
charides with 3H-labeled D-glucosaminitol at the reducing
ends may yield several labeled products, as discussed by other
investigators (43, 46). Acid hydrolysis of 3H-reduced degra-
dation products in 4
N
HC1 at 100
"C
for
8
h yielded only a
small amount of 3H-labeled D-glucosaminitol (3.4% of total
tritium). These monosaccharides appeared to be derived from
the original reducing termini of intact
HS
rather than from
the reducing termini of degradation fragments, since it was
found that most of the reduced and acid-hydrolyzed products
from the original reducing terminal saccharides were D-glu-
cosaminitol with smaller amounts of D-galactitol and D-xyli-
tol, and the first borohydride reduction of HS was not com-
plete in
0.1
M
borate buffer, pH
8.0.
Thus, the HS-degrading
endoglycosidase (heparanase) of B16 cells cleaves HS at the
glucuronidic linkages.
B16 melanoma cells are known to produce S-GAGS such as
by guest, on July 13, 2011www.jbc.orgDownloaded from
Metastatic
Tumor
Cell Heparanme
2289
chondroitin sulfate and HS
(47, 48).
In our experiments the
amount of 3H-reduced monosaccharides derived from endog-
enous
S-GAG
fragments in the incubated cell extract itself
was negligible, compared to that from the degradation prod-
ucts of the exogenous substrate HS.
DISCUSSION
Metastatic
B16
melanoma cells must invade both the vas-
cular endothelial cell layer and the underlying basal lamina,
solubilizing matrix components such
as
glycoproteins and
S-
GAGs (6, 17-19, 24)
in order to accomplish the blood-borne
metastasis. We have studied the abilities of metastatic
B16
cells to cleave
GAGs,
important portions of the endothelial
matrix proteoglycans, and have found a
B16
cell, HS-specific
endoglucuronidase. Of the seven types of
GAGs
tested, only
HS was degraded at high rates by
B16
melanoma cell extracts.
This is consistent with previous findings that HS is a promi-
nent component of the
B16
cell-solubilized subendothelial
basal lamina-like extracellular matrix
(17).
HS is thought to be an important structural component of
basal laminae, and its localization in a variety of tissues has
been demonstrated using cationic molecules, dyes
(49-53)
or
antibodies
(54,55).
Laurie
et
al.
(55)
have shown that type IV
collagen, laminin, HS-proteoglycan, and fibronectin are lo-
calized in the basal lamina by using antibodies directed
against each of these components. HS, but not other
GAGs
in the glomerular basement membrane, appears to serve as a
permeability barrier against macromolecules
(56).
In these
and other studies on basal laminae
(17, 51, 54, 57, 58),
HS or
HS-proteoglycan was the major
S-GAG
component. HS may
be involved in basal lamina matrix assembly by promoting
the interactions of collagenous and noncollagenous protein
components and inhibiting proteolytic activity
(59).
HS-proteoglycan in the vascular endothelial basal lamina
is thought to act as a barrier against malignant cell invasion.
Thus, degradation
of
HS may be essential for malignant cell
invasion through the vascular wall. We have found that highly
metastatic
B16
melanoma cells solubilize
HS
in the suben-
dothelial matrix as well as degrade purified soluble HS
(6, 17-
19).
It is of interest that
B16
sublines with higher lung-
colonizing abilities have higher HS endoglycosidase activities
(18),
although
B16
sublines with different metastatic poten-
tials possess similar HS exoglycosidase activities as suggested
from the results of HS degradation experiments in the absence
or presence of P-glucuronidase inhibitor. These results are
compatible with the data of Sloane
et
al.
(22,
23),
who failed
to find significant differences in lysosomal &glucuronidase
activities between
B16
sublines.
HS-degrading endoglycosidases have been found in various
tissues such as human skin fibroblasts
(60),
rat liver cells
(61),
human placenta
(25),
and human platelets
(26, 46,
62,
63).
Klein and von Figura
(25)
partially purified a HS-specific
endoglucuronidase from human placenta, and Oosta
et al.
(63)
have purified a platelet endoglucuronidase that degrades both
heparin and HS. The physiological roles of these enzymes
have not yet been elucidated, although Wasteson
et al.
(26)
have postulated that the platelet enzyme may be involved in
modifying endothelial cell surfaces influencing the functional
properties
of
the vascular lining, and Castellot
et al.
(64)
have
proposed a possible role for the platelet enzyme in modulating
the proliferation of vascular smooth muscle cells.
We found that a HS-specific endoglycosidase in
B16
mela-
noma cells was an endoglucuronidase. HS-degrading endog-
lycosidases in mammalian cells reported previously by other
investigators were called “heparitinases” to indicate heparitin
sulfate (heparan sulfate)-specific endoglycosidase. However,
heparitinase originally was used to designate an elimination
enzyme
(EC 4.2.2.8)
in
Flavobacterium heparinum,
and this
enzyme cleaves nonsulfated and monosulfated 2-acetoamido-
2-deoxy-c~-~-glucosy1-D-hexuronic
acid linkages of HS
(65-
68).
Since HS-specific endoglycosidases in mammalian cells
are endoglucuronidases, except for one found in skin fibro-
blasts
(60),
mammalian cell endoglucuronidases capable of
degrading HS should be called “heparanases” consistent with
the currently used term heparan sulfate.
The pH optima of
B16
heparanase in crude extract was
5-
6;
however, intact cells showed high heparanase activities at
physiological pH
(18).
In our experiments HS degradation by
B16
heparanase was inhibited by heparin. Similar observa-
tions on the platelet enzyme were reported by Wasteson
et
al.
(26);
therefore, certain properties of these two enzymes appear
to be similar. However, their substrate specificities were dif-
ferent. The platelet enzyme depolymerizes both HS and hep-
arin into small oligosaccharides
(26,46, 62, 63).
On the other
hand,
B16
heparanase cleaved specific portions of the HS
chain, producing characteristically large polysaccharide frag-
ments. In the presence of SAL,
B16
heparanase was very
active against HS, while heparin was not cleaved at significant
rates, even after prolonged incubation. This substrate speci-
ficity may be restricted by carbohydrate sequence but not by
molecular size, since
B16
melanoma cell heparanase degraded
HS from bovine lung
(M,
-
34,000)
(18),
BAE
matrix
(Mr
-
24,000),
BCE
matrix
(Mr
- 48,000),’ PYS-2
carcinoma cell
(Mr
-
11,000) (281,
and
EHS
sarcoma tissue
(M,
-
70,000)
into characteristic
M,
fragments, while heparin from hog
intestinal mucosa
(M,
-
11,000)
or bovine lung
(M,
- 15,000)
was not degraded
at
significant rates.
Ogren and Lindahl
(43)
reported that a murine mastocy-
toma possesses a heparin-specific endoglucuronidase capable
of converting macromolecular branched heparin to physiolog-
ically active intermediate molecular weight fragments. The
cleavage of heparin by mastocytoma heparinase occurs in the
N-acetyl-D-glucosamine-rich
regions of the heparin chain
(43).
In the cleavage of HS by
B16
heparanase, the recognition
of N-acetyl groups as well as sulfate groups may be important,
since this enzyme can distinguish HS from heparin and
cleaves HS at specific intrachain sites. In general,
HS
differs
from heparin by its higher content of D-glucuronic acid resi-
dues and N-acetyl groups and lower sulfate
(69).
HS from
various sources can be further subfractionated by the hetero-
geneity of
N-
and 0-sulfation, and this heterogeneity may be
tissue- and transformation-dependent
(40,69-74).
Linker and
Hovingh
(69)
described the structure of
HS
as “block-type”
with areas free of or low in sulfate, high in N-acetyl groups,
and intermediate or high in
0-
and N-sulfate.
B16
heparanase
may recognize one or more of these block-type structures.
When
B16
heparanase attacked HS from subendothelial ma-
trix, the intermediate-sized fragments were produced
(17).
However, the average
M,
of HS degradation fragments from
BAE
subendothelial matrix was larger than the average
M,
of
HS
degradation fragments from other sources. Interestingly,
HS produced by cultured vascular endothelial cells has been
reported to have higher N-sulfate content than HS from other
sources
(75, 76).
These findings may provide an explanation
as to why HS in the endothelial extracellular matrix was
degraded into large fragments. Because of its block-type struc-
ture of low N-acetyl group content (heparin-like structure),
vascular endothelial matrix HS may have only a few linkages
susceptible to
B16
heparanase.
Subcellular fractionation studies by Oosta
et
al.
(63)
indi-
cate that the platelet enzyme is present within lysosomes.
B16
heparanase may also be localized in lysosomes.
B16
by guest, on July 13, 2011www.jbc.orgDownloaded from
2290
Metastatic
Tumor
Cell
Heparanase
heparanase has been found in serum-free culture media
of
B16
cells3 as well as in plasma membrane vesicles sponta-
neously shed from the melanoma cells (77). When
HS
degra-
dation by intact viable cells was examined, the differences in
heparanase activity among
B16
sublines were more dramatic
(18),
suggesting that heparanase localization and its mecha-
nism of release may be important for the
in
vivo
properties of
malignant cells. Liotta
et
al.
(21,
78) reported that metastatic
tumor cells released type
IV
collagen-specific neutral protease,
which correlated with metastatic potential, and Sloane
et
al.
(79) reported that cathepsin
B,
a lysosomal cystein proteinase
correlating with
B16
metastatic potential
(22,
23),
was also
cell released. It is likely that metastatic cells require hepar-
anase as well as type
IV
collagenase and cathepsin
B
for
tumor cell invasion through the vascular endothelial basal
lamina.
Acknowledgments-We wish to thank Adele Brodginski and
Eleanor Felonia for their assistance in the preparation
of
this man-
uscript.
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by guest, on July 13, 2011www.jbc.orgDownloaded from
... However, three years earlier in 1975, a heparanase-like endoglucuronidase from murine mast cells was found able to cleave macromolecular heparin to functional heparin [22] and in 1983 lymphoma cells were found able to degrade HS chains of HSPGs of subendothelial ECM [23]. Finally, in 1984 other authors reported for the first time in the title of their paper the term heparanase to design an endoglucuronidase, able to degrade HS, produced by the highly invasive lung metastatic murine B16-BL6 melanoma cells [24]. Endoglucuronidase activities were previously described in other mammalian tissues and cells such as rat liver tissues [25], human skin fibroblasts and placenta [26], human platelets [27] and activated T lymphocytes [28]. ...
... Hence, heparanase localization and activating processes are relevant in determining its biological function in a variety of healthy and malignant cells and tissues [35]. Moreover, heparanase upregulation has been described in several malignancies and pathological conditions including acute and chronic inflammation, fibrosis, amyloidosis, diabetes and related nephropathies, osteoarthritis, atherosclerosis and other vessel wall pathologies [3,6] The first evidence of heparanase activity in the murine melanoma B16-BL6 [24] and T-lymphoma [23] experimental models, were provided by Nakajima et al. [24] and Vlodavsky et al. [23] associating the in vivo metastatic potential of these cells with HS degradation. Excellent reviews [2,3,6,7,11] reported findings on heparanase overexpression in several malignancies and functional studies in cancer models highlighting its causal relevant role in sustaining tumor growth and progression. ...
... Hence, heparanase localization and activating processes are relevant in determining its biological function in a variety of healthy and malignant cells and tissues [35]. Moreover, heparanase upregulation has been described in several malignancies and pathological conditions including acute and chronic inflammation, fibrosis, amyloidosis, diabetes and related nephropathies, osteoarthritis, atherosclerosis and other vessel wall pathologies [3,6] The first evidence of heparanase activity in the murine melanoma B16-BL6 [24] and T-lymphoma [23] experimental models, were provided by Nakajima et al. [24] and Vlodavsky et al. [23] associating the in vivo metastatic potential of these cells with HS degradation. Excellent reviews [2,3,6,7,11] reported findings on heparanase overexpression in several malignancies and functional studies in cancer models highlighting its causal relevant role in sustaining tumor growth and progression. ...
Chapter
The chapter will review early and more recent seminal contributions to the discovery and characterization of heparanase and non-anticoagulant heparins inhibiting its peculiar enzymatic activity. Indeed, heparanase displays a unique versatility in degrading heparan sulfate chains of several proteoglycans expressed in all mammalian cells. This endo-β-D-glucuronidase is overexpressed in cancer, inflammation, diabetes, atherosclerosis, nephropathies and other pathologies. Starting from known low- or non-anticoagulant heparins, the search for heparanase inhibitors evolved focusing on structure-activity relationship studies and taking advantage of new chemical-physical analytical methods which have allowed characterization and sequencing of polysaccharide chains. New methods to screen heparanase inhibitors and to evaluate their mechanism of action and in vivo activity in experimental models prompted their development. New non-anticoagulant heparin derivatives endowed with anti-heparanase activity are reported. Some leads are under clinical evaluation in the oncology field (e.g., acute myeloid leukemia, multiple myeloma, pancreatic carcinoma) and in other pathological conditions (e.g., sickle cell disease, malaria, labor arrest).
... Experimental heparanase overexpression in a transgenic mouse model verified extensive HS fragmentation in vivo (37). The fragmentation of HS chains was the first appreciated function of this protein, and it remains the key heparanase function assayed by the majority of drug discovery programs targeting heparanase (5,20,38). The degradation of HS chains on matrix proteoglycans by heparanase, as well as dissolving the physical barrier, also releases latent growth factors, like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), and keratinocyte growth factor (FGF4) which are bound and sequestered to the matrix by HS (20). ...
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Heparanase has been viewed as a promising anti-cancer drug target for almost two decades, but no anti-heparanase therapy has yet reached the clinic. This endoglycosidase is highly expressed in a variety of malignancies, and its high expression is associated with greater tumor size, more metastases, and a poor prognosis. It was first described as an enzyme cleaving heparan sulfate chains of proteoglycans located in extracellular matrices and on cell surfaces, but this is not its only function. It is a multi-functional protein with activities that are enzymatic and non-enzymatic and which take place both outside of the cell and intracellularly. Knowledge of the crystal structure of heparanase has assisted the interpretation of earlier structure-function studies as well as in the design of potential anti-heparanase agents. This review re-examines the various functions of heparanase in light of the structural data. The functions of the heparanase variant, T5, and structure and functions of heparanase-2 are also examined as these heparanase related, but non-enzymatic, proteins are likely to influence the in vivo efficacy of anti-heparanase drugs. The anti-heparanase drugs currently under development predominately focus on inhibiting the enzymatic activity of heparanase, which, in the absence of inhibitors with high clinical efficacy, prompts a discussion of whether this is the best approach. The diversity of outcomes attributed to heparanase and the difficulties of unequivocally determining which of these are due to its enzymatic activity is also discussed and leads us to the conclusion that heparanase is a valid, but challenging drug target for cancer.
... The only mammalian endoglycosidase capable of degrading HS is known as heparanase (HPSE); when released into the ECM, HPSE degrades HS cleaving polymeric HS molecules; additionally, this ubiquitously expressed enzyme is involved in a number of intracellular processes including autophagy, endocytosis, gene transcription, and secretion of exosomes [2][3][4]. HPSE is an endo-β-glucuronidase of the Carbohydrate Active Enzymes (CAZy) Glycoside Hydrolase (GH)79 family. ...
... The sole mammalian HS-degrading enzyme, heparanase, is secreted mainly by tumor cells and leukocytes as a latent 65 kDa pro-enzyme, which is then internalized and activated by cathepsin L-mediated proteolytic cleavage to form an active heterodimer composed of the 50 kDa and 8 kDa polypeptides 21 . Through its endo-b-glucuronidase activity, heparanase cleaves the HS side-chains of proteoglycans thereby releasing 4e7 kDa fragments of HS that can remain biologically active 22 , but it does not degrade hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate or keratan sulfate 23 . Heparanase can release HS-bound growth factors, cytokines, chemokines and other ligands stored within the ECM, some of which have also been implicated in cartilage metabolism 24e27 . ...
Article
Objectives: The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. Methods: Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative PCR. MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. ERK phosphorylation was evaluated by Western blot. Human OA cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. Results: Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by FGF2 neutralizing antibodies and by a FGF-receptor inhibitor. Conclusions: Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.
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The glycosaminoglycans (GAGs) on cell surfaces play significant roles during cancer development, and the heparanase activity is strongly implicated in the structural remodeling of the extracellular GAG matrix, potentially leading to tumour cell invasion. Polymer-protein/peptide conjugates are one of the most promising approaches for anticancer therapy due to their controllability, biocompatibility, and targeting properties. In this study, distinct and well-defined glycopolymer-peptide conjugates, mimicking the multivalent architecture found in GAGs, were synthesised for targeting and killing tumour cells. Regio-selectively sulphated galactosamine derivatives were chemically synthesised, and six GAGs-mimetic glycopolymers were generated by post-modification based on the ring-opening metathesis polymerization (ROMP). The glycopolymers with diverse galactosamine sulphation patterns showed significant inhibitory effects on heparanase. Glycopolymers decorated with 3,4,6-O-sulphated GalNAc exhibited the highest activities, inhibiting heparanase as well as tumour cell proliferation. We demonstrated that a novel glycopeptide mimetic, end-functional conjugation of iRGD peptide on glycopolymer, could effectively enter HeLa cells and inhibit signalling pathways involved in tumour cell proliferation. These findings should promote the development of novel glycomimetics for specific tumour-targeted therapies.
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In 2019, we mark the 20th anniversary of the cloning of the human heparanase gene. Heparanase remains the only known enzyme to cleave heparan sulfate, which is an abundant component of the extracellular matrix. Thus, elucidating the mechanisms underlying heparanase expression and activity is critical to understanding its role in healthy and pathological settings. This chapter provides a historical account of the race to clone the human heparanase gene, describes the intracellular and extracellular function of the enzyme, and explores the various mechanisms regulating heparanase expression and activity at the gene, transcript, and protein level.
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In this chapter, we will emphasize the importance of heparan sulfate proteoglycans (HSPG) in controlling various physiological and pathological molecular mechanisms and discuss how the heparanase enzyme can modulate the effects triggered by HSPG. Additionally, we will also navigate about the existing knowledge of the possible role of heparanase-2 in biological events. Heparan sulfate is widely distributed and evolutionarily conserved, evidencing its vital importance in cell development and functions such as cell proliferation, migration, adhesion, differentiation, and angiogenesis. During remodeling of the extracellular matrix, the breakdown of heparan sulfate by heparanase results in the release of molecules containing anchored glycosaminoglycan chains of great interest in heparanase-mediated cell signaling pathways in various physiological states, tumor development, inflammation, and other diseases. Taken together, it appears that heparanase plays a key role in the maintenance of the pathology of cancer and inflammatory diseases and is a potential target for anti-cancer therapies. Therefore, heparanase inhibitors are currently being examined in clinical trials as novel cancer therapeutics. Heparanase-2 has no enzymatic activity, displays higher affinity for heparan sulfate and the coding region alignment shows 40% identity with the heparanase gene. Heparanase-2 plays an important role in embryogenic development however its mode of action and biological function remain to be elucidated. Heparanase-2 functions as an inhibitor of the heparanase-1 enzyme and also inhibits neovascularization mediated by VEGF. The HPSE2 gene is repressed by the Polycomb complex, together suggesting a role as a tumor suppressor.
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Two decades following the cloning of the heparanase gene, the significance of this enzyme for tumor growth and metastasis cannot be ignored. Compelling pre-clinical and clinical evidence tie heparanase with all steps of tumor formation namely, initiation, growth, metastasis, and chemo resistance, thus confirming and significantly expanding earlier observations that coupled heparanase activity with the metastatic capacity of tumor cells. This collective effort has turned heparanase from an obscure enzyme to a valid target for the development of anti-cancer drugs, and led basic researchers and biotech companies to develop heparanase inhibitors as anti-cancer therapeutics, some of which are currently examined clinically. As expected, the intense research effort devoted to understanding the biology of heparanase significantly expanded the functional repertoire of this enzyme, but some principle questions are still left unanswered or are controversial. For example, many publications describe increased heparanase levels in human tumors, but the mechanism underlying heparanase induction is not sufficiently understood. Moreover, heparanase is hardly found to be increased in many studies utilizing methodologies (i.e., gene arrays) that compare tumors vs (adjacent) normal tissue. The finding that heparanase exert also enzymatic activity-independent function significantly expands the mode by which heparanase can function outside, but also inside the cell. Signaling aspects, and a role of heparanase in modulating autophagy are possibly as important as its enzymatic aspect, but these properties are not targeted by heparanase inhibitors, possibly compromising their efficacy. This Book chapter review heparanase function in oncology, suggesting a somewhat different interpretation of the results.
Article
We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.
Article
Objective: To investigate the effect of unfractionated heparin on the expression of serum and liver tissue heparanase (HPA) in mice with liver injury induced by sepsis. Methods: Forty-eight healthy male C57BL/6 mice aged 6-8 weeks were divided into groups according to random number table method. Twenty-four septic mice models (CLP group) were established by cecal ligation and puncture (CLP); the other 24 mice underwent sham operation (sham group), only laparotomy and abdominal closure were performed without ligation. Twelve mice in sham group and CLP group received heparin pretreatment (sham+UFH group, CLP+UFH group), and 8 U heparin unfractionated heparin (diluted to 200 μL) was injected into the tail vein of the mice at 30 minutes and 12 hours after operation respectively. The other 12 mice were injected with the same amount of normal saline. The serum and liver tissues of mice were collected at 4 and 24 hours after CLP. The levels of serum HPA, interleukin (IL-6, IL-1β), tumor necrosis factor-α (TNF-α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by enzyme linked immunosorbent assay (ELISA). The pathological changes of liver tissue were observed with hematoxylin eosin (HE) staining. The expression of HPA in liver tissue was detected by immunohistochemistry. Results: Compared with the sham group, the levels of serum HPA, IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were increased significantly, and increased further over time. The histopathology examination was performed, and abnormal structure, inflammatory cell infiltration, liver cell necrosis could be found in the tissue. The expression level of HPA in liver tissue was detected by immunohistochemistry, which was increased after CLP. This indicated that the animal model of sepsis was successfully prepared. Compared with CLP group, serum HPA, inflammatory factors and transaminase levels were significantly decreased at 4 hours after operation in group CLP+UFH [HPA (ng/L): 76.72±2.75 vs. 101.55±7.54, IL-6 (ng/L): 51.16±5.68 vs. 63.89±3.26, IL-1β (ng/L): 31.53±2.90 vs. 40.87±2.88,TNF-α (ng/L): 171.76±5.60 vs. 194.62±14.13, ALT (μg/L): 0.26±0.09 vs. 0.62±0.17, AST (μg/L): 1.03±0.22 vs. 1.45±0.08, all P < 0.05]. At 24 hours, it was significantly higher than that of 4 hours, but they were significantly lower than those in CLP group [HPA (ng/L): 125.30±7.80 vs. 302.50±17.81, IL-6 (ng/L): 81.16±4.54 vs. 176.56±5.45, IL-1β (ng/L): 61.13±2.80 vs. 113.73±3.96, TNF-α (ng/L): 328.47±10.79 vs. 599.62±10.20, ALT (μg/L): 0.38±0.17 vs. 0.91±0.26, AST (μg/L): 1.16±0.15 vs. 1.88±0.08, all P < 0.05]. It was shown by HE staining that the edema of liver tissue decreased and inflammatory cell infiltration decreased. It was shown by immunohistochemistry that the expression level of HPA in liver tissue was significantly decreased [A value (×10-3): 2.49±0.93 vs. 6.05±1.22 at 4 hours, 1.86±0.77 vs. 7.55±0.35 at 24 hours, both P < 0.05]. There was no significant difference in indexes between the sham+UFH group and the sham group. Conclusions: The expression of HPA was significantly increased during sepsis in mice. Unfractionated heparin may mitigate liver injury by inhibiting HPA.
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Crude enzyme obtained from heparin-induced flavobacteria has been fractionated into a heparitinase acting on heparitin sulfates and related compounds and a heparinase acting mainly on heparin. Purification achieved for each was from 50 to 100 times that of earlier preparations containing a mixture of the two enzymes. In agreement with previous data both enzymes act as eliminases rather than hydrolases yielding products containing Δ4,5-unsaturated uronic acid. Specificity of the heparitinase appears to require the absence of O-sulfate and the presence of N-acetyl or sulfamido groups. Specificity of the heparinase requires the presence of O-sulfate and sulfamido groups while derivatives containing free amino or N-acetyl are not substrates. The heparinase degrades heparitin sulfate to some extent acting apparently on the heparin-like portion.
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An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.
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Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-microns thick slices of formaldehyde-fixed tissues. In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries. The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.
Article
Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.
Article
The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.
Article
Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan-sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.
Article
Several commerical batches of heparitin sulfate extracted from beef lung tissue were fractionated into at least four distinct mucopolysaccharides by a combination of polyacrylamide and agarose gel electrophoresis. The four heparitin sulfates (A, B, C and D) were distinguished from each other and from heparin by several physical and chemical properties such as electrophoretic migration, molecular weight, presence of N-acetyl, N- and )-sulfate residues, optical rotation and enzymatic degradation. Of particular significance was the isolation of a heparitin sulfate (heparitin sulfate C) with a homogeneous molecular weight.