J. Embryol. exp. Morph. Vol. 31, 2, pp. 319-328, 1974
Printed in Great Britain
319
Biochemical observations on the cartilage of
achondroplastic (can) mice
By D. R. JOHNSON 1 AND D. M. HUNT 2
Department of Animal Genetics, University College London
SUMMARY
The achondroplastic can/can embryo can be distinguished from normal litter-mates at
17 days by reduced protein synthesis. After 3 days post parturn protein synthesis is increased
to levels well above normal, as is incorporation of glucose into mucopolysaccharides and the
levels of uridine diphosphoglucose dehydrogenase and UDP-glucose-4-epimerase.
INTRODUCTION
Although achondroplasia is a common hereditary defect in man, domestic
and laboratory animals few investigations exist at other than the gross histological level. Notable exceptions to this are the electron microscope studies on
the achondroplastic (ac) rabbit (Shepard, Fry & Moffett, 1969) and the chondrodystrophic (cho) and cartilage anomaly (can) mouse (Johnson & Wise, 1971;
Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon, 1972).
The achondroplastic rabbit and the nannomelic chick have also been studied
using biochemical techniques (Fraser & Goetinck, 1971; Shepard & Bass, 1971;
Bargman, Mackler & Shepard, 1972). In man cartilage from seven dwarf
conditions was examined microchemically by Stanescu, Stanescu & Szirmai
(1972).
These contributions suggest that the underlying causes of achondroplasia
may be very varied. Seegmiller has identified aberrant cross-banded collagen in
the cho mouse; Fraser & Goetinck have noted a decreased uptake of radioactive mucopolysaccharide precursors in the nannomelic chick, whilst the basis
of achondroplasia in the ac rabbit seems to be a deficiency in a mitochondrial
enzyme.
Because of the paucity of information and because of the diversity of that
which exists it was decided to follow up the previous ultrastructural study of the
cartilage anomaly mouse by a biochemical investigation.
1
Author's address: Department of Animal Genetics, Wolfson House, Stephenson Way,
London NW1 2 HE, U.K.
2
Author's address: Department of Genetics, University of Glasgow, Church St., Glasgow
W1,U.K.
320
D. R. JOHNSON AND D. M. HUNT
MATERIAL AND METHODS
Cartilage was isolated from the sternum and uncalcified ribs of can/can mice
and normal litter-mates aged 0-5 days. The ribcage was removed by lateral
incisions and the ossified portion of the ribs cut off. Musculature was removed
by gentle scraping with a scalpel and the individual cartilaginous ribs dissected
free and cut into pieces ca. 0-3 mm long. The pieces were then weighed and
incubated for 2, 3 or 4 h in a shaking water-bath in Waymouth's solution, to
which had been added 100 i.u. each of streptomycin and penicillin and appropriate trace amounts of labelled precursors ([l-14C]D-glucosamine, 55 mCi/
mmol; [l-14C]D-galactosamine, 58mCi/mmol; [35S]sodium sulphate, 100 mCi/
mmol; [U-14C]glucose, 285mCi/mmol; Radiochemical Centre, Amersham).
The supernatant medium was discarded and the tissue washed in 2 x 1 ml cold
Waymouth's solution and homogenized in 0-6 ml/mg distilled water. Proteinbound radioactivity was isolated by precipitation with an equal volume of 10 %
trichloracetic acid (TCA) followed by centrifugation and two washes in 5 %
TCA. Bovine serum albumin (0-0005 g/ml) was added as carrier protein. The
precipitate was redissolved in 3 N - N H 4 O H and counted in Unisolve (KochLight Ltd.) in a Packard Tricarb liquid scintillation spectrophotometer with
external standardization.
Mucopolysaccharide-bound label was measured after digesting protein with
papain. Crude papain (0-01 g/ml) was dissolved in 0-1 M-phosphate buffer
containing 0-005M-EDTA and 0-005 M-cysteine, and added to the cartilage
homogenate. Digestion was allowed to proceed overnight at 70 °C. All mucopolysaccharides were precipitated with 1 % cetylpyridinium chloride (CPC)/
0-04M-NaCl; hyaluronic acid and chondroitin sulphates were redissolved in
0-1 % CPC/0-4M-NaCl, 0-1 % CPC/l-2M-NaCl respectively and counted in
Unisolve.
The action of chondroitinase ABC (Seikaguko Kogyo Co., Tokyo) on
protein-bound radioactivity was estimated by incubating labelled cartilage
homogenate with chondroitinase ABC in 0-125M Tris/HCl buffer + 0-075 Msodium acetate (pH 8-0) for 60 min at 37 °C. The reaction was stopped with an
equal volume of 10 % TCA.
Activity of the enzymes glucose-6-phosphate dehydrogenase, uridine diphospho (UDP)-glucose dehydrogenase and UDP-glucose-4-epimerase was
assayed fluorimetrically. Cartilage was homogenized in an appropriate buffer
(0-lM-Tris, pH 7-6, 0-1 M-glycine, pH 8-7, 0-1 M-glycine, pH 8-7) and centrifuged
at 10 000 g for 20 min. An aliquot of the supernatant was incubated for 15 min
at 37 °C with appropriate substrate (0-025M-glucose-6-phosphate, 0 - 0 2 M - U D P glucose, 0-7 mM UDP-galactose plus UDPG-dehydrogenase plus 0 - 0 3 M - N A D ) .
The formation of NADH was measured at 340rn.fiexcitation, 460 m/i emission
using a standard containing 0-2 ^mole/ml NADH. Standards and blanks also
contained boiled cartilage supernatant.
Biochemistry 0/can cartilage
321
Oxidative phosphorylation at site III of liver mitochondria was assayed using
Bargman, Mackler & Shepard's (1972) modification of the technique of Sandai
& Jacobs (1960): the reaction was performed in a test tube rather than a Warburg apparatus, and esterified phosphorus only measured.
Protein synthesis was measured by incubating cartilage or liver with [U-14C]glycine (114mCi/mmol) as previously described. After washing, the tissue was
homogenized in 0-lM-Tris buffer (pH 7-2) containing 0-lM-CaAc. An aliquot
was incubated with 100 units of collagenase (Sigma Biochemicals, type III) for
30 min at 37 °C. Remaining protein was precipitated with TCA and counted as
described above. The collagenase was shown to be protease-free by repeating the
above experiment with [U-14C]tryptophan (45 mCi/mmol) as substrate. The
collagenase failed to remove any counts in this experiment.
Litters of 17- and 18-day-old embryos were obtained by priming female
offspring from matings segregating for can (f of which are +lcari) with
3 i.u. serum gonadotrophin followed after 40 h by 3 i.u. chorionic gonadotrophin (Folligon and Chorulon, Organon Labs.). The females were then
mated to known +/can $$. Cartilage preparations from 17- and 18-day-old
embryos were made as described above.
RESULTS
The cartilaginous matrix has as its main components collagen and mucopolysaccharides. The mucopolysaccharides are attached to a protein backbone
after synthesis. Techniques for the estimation of polysaccharides fall into two
groups, those which degrade the protein backbone and measure total polysaccharides and those which precipitate the protein-bound polysaccharides and
disregard the remainder.
In the neonatal rat epiphysis (Handley & Phelps, 1972) the mucopolysaccharides are of two main kinds, hyaluronic acid (4 %), a repeating polymer
of glucuronic acid linked to JV-acetylglucosamine, which is unsulphated and
contains no galactose, and the chondroitin sulphates (chondroitin 4-sulphate
65 %, chondroitin 6-sulphate 15 %) which have alternating glucuronic acid and
iV-acetylgalactosamine residues and are sulphated. Keratan sulphate (2 %) is
also present.
Protein-bound radioactivity is incorporated at identical rates by + and
can/can newborn cartilage, whether the precursor is glucosamine, galactosamine
or sulphate (Table 1). At three days the incorporation of glucosamine residues is
significantly higher in can/can than in + ; this has returned to normal by 5 days.
Incorporation of galactosamine and sulphate is normal throughout.
The increase in glucosamine is due to increased uptake rather than increased
turnover: in a chase experiment (Table 2) there was no increased breakdown in
can/can mucopolysaccharides in the 2 h period during which the tissue was
exposed to unlabelled medium. The increased incorporation represents increased
322
D . R. J O H N S O N A N D D . M. H U N T
Table 1. Incorporation of protein-bound radioactive precursors
into + and can/can cartilage
+
can/can
+
can/can
+
can/can
[l- C]D-glucosamine
-14C]D-galactosamine
[35S]sulphate
5 days
3 days
Newborn
14
2-69 ±0-54 (6)
3-25 ±0-34 (6)
5-92 ±0-39 (4)
2-06±0-52 (5)
6-47±0-39 (5)*** 603±0-39 (4)
0-395±0045 (4) 0-349±0024 (4)
—
0340 ± 0 0 4 3 (4) 0-313 ±0-021 (4)
—
1 -60±0-24 (8)
1 -32±005 (9)
1 -32±008 (4)
1 • 18 ± 0-22 (7)
1 -27 ± 005 (8)
1 -29 ± 0-13 (4)
Results in nmol/g/h ± S.E.M. Figures in brackets indicate number of determinations.
*** P < 0 0 1 .
Table 2. Incorporation of [l-uC]D-glucosamine into can/can and
normal cartilage at 3 days old. Chase experiment
Normal
can/can
2h
2h + 2h
chase
4h
2h
2h + 2h
chase
4h
5-37
4-72
703
4-88
13-26
10-30
10-68
7-68
11-25
8-61
18-34
12-53
A
1
Results in nmol/g.
Table 3. Effect of chondroitinase ABC on incorporated
[\-x*C]D-glucosamine at 3 days old
Normal
r
can/can
V
Control
+ ABC
/o
reduction
Control
+ ABC
V
/o
reduction
620
862
1468
720
266
295
555
279
57-1
65-7
62-2
61-2
1244
1084
1353
1051
498
383
471
376
600
64-7
65-2
64-2
Results are in dpm/mg/3 h incubation.
synthesis of mucopolysaccharides susceptible to the action of chondroitinase
ABC (Table 3).
Non-protein-bound radioactivity follows a similar but distinct pattern
(Table 4). At 3 days the incorporation from glucosamine into hyaluronic acid is
significantly raised, and that into chondroitin sulphate increased, but not
significantly so. At 5 days both fractions contain significantly more 14C label.
The incorporation of 35S is normal throughout, although very variable in 3-day
canjcan chondroitin sulphate. Trace amounts of sulphate label (< 5 % of the
Biochemistry o/can cartilage
323
Table 4. Incorporation of label into mucopolysaccharide fractions
of + and can/can cartilage
Hyaluronic acid fraction
35S
can/can
Chondroitin sulphate fraction
14 C
35
S
14
C
35 S
can/can
14
C
35 S
3 days
5 days
19-81 ±10(4)
1-42 ±01 (4)
33-55 ±4-8 (4)**
0-98 ±0-3 (4)
72-85 ±22-0 (4)
41-92 ±7-7 (4)
98-78 ±23-6 (3)
83-92 ±33-4 (3)
30-59 ±5-4 (8)
6-76±10(8)
58-37 ±9-6 (8)*
7-10 ±2-2 (7)
151-2± 19-7 (5)
135-6 ±21 -4 (6)
287-3 ±21-4 (7)*
136-7 ±12-5 (8)
Cartilage was incubated for 2 h with 1 /tCi/ml label, deproteinized with papain, and mucopolysaccharide fractions differentially precipitated with cetylpyridinium chloride. Results in
3
dpm/g/hx 10 ±S.E.M.
* P = < 005,
** P = < 002.
Table 5. Incorporation of label into mucopolysaccharides of
+ and can/can skin at 3 days old
Hyaluronic acid fraction
+
can/can
11-41 ±1-35 (4)
502 ±111 (4)
1015 ±106 (3)
4-38 ±0-87 (3)
2-66±005 (3)
1-49 ±0-21 (3)
2-43 ±0-42 (4)
1-32 ±0-27 (4)
Chondroitin sulphate fraction
Details as in Table 4.
Table 6. Activity of some enzyme systems in + and can/can cartilage
Newborn
3 days
5 days
0167±0015 (7) 0156 ±0013 (5) 0-165 ±0-004 (5)
UDPG-dehydrogenase
can/can 0183±0017 (6) 0148±0012 (4) 0-227±0044 (4)**
0139±0010 (5) 0152±0004 (5) 0109±0003 (5)
U DPG-4-epimerase
can/can 0147 ±0011 (4) 0129±0025 (4) 0-205±0020 (4)***
G-6-P dehydrogenase
can/can
0021 ±0001 (5) 0035±0002 (4)
0020 ±0001 (4) 0033 ± 0001 (4)
Results in/tmol NADH formed per 15 min per 001 g cartilage±S.E.M.
** p = < 002, *** P = < 001.
total) were also found in the hyaluronic acid fraction, presumably as a contaminant.
This increase in 14C incorporation is limited to cartilage. Samples of + and
can/can skin treated in a similar way showed no such difference (Table 5). It
should be noted that the 'hyaluronic acid' fraction of skin contained large
amounts of 35S label. This is thought to be due to the wide range of acid muco-
324
D. R. JOHNSON AND D. M. HUNT
Table 7. Incorporation of [uC]glycine into + and
can/can cartilage and liver protein
Cartilage
+
can/can
+
can/can
Liver
Newborn
3 days
5 days
18-72± 1-21 (12)
14-28 ± 119 (6)**
3-21 ±0-27 (6)
2-46 + 0-44(5)
22-42±212 (5)
13-51 ±1-89 (4)**
305±0-37 (5)
3-32 ±0-56 (4)
26-66± 1-79 (5)
44-72±6-56 (3)**
3-52±0-29 (5)
3-50 ±0-50 (3)
Results in dpm/10mg cartilage/2hx lO"3.
** P = < 002.
Table 8. Effect of collagenase on [uC]glycine incorporated into
+ and can/can cartilage protein
3 days
5 days
+
Total incorporation
Collagenase soluble
Collagenase insoluble
can/can
Total incorporation
Collagenase soluble
Collagenase insoluble
14-77 ± 0-98 (8)
864±0-81 (8)
6-13 ±0-46 (8)
26-64 + 1-12 (5)
15-33±0-70 (5)
11-37 ±0-52 (5)
1014±0-50 (4)**
1-89±0-43 (4)**
8-25 ±0-44 (4)**
57-46±514 (4)**
28-89±l-25 (4)**
26-57 ± 1-40 (4)**
Results in dpm/10 mg/2hx 10~ 3 ±S.E.M. Cartilage samples were incubated for 2h with
1 fbCi [14C]glycine then aliquots containing 10 mg cartilage were exposed to 100 u. collagenase
for 30min at 37 °C.
** P = <002.
polysaccharides found in skin (Hardingham & Phelps, 1968, 1970a, b) which
were not fully resolved by the technique used.
Two cartilage-specific enzyme systems, UDP-glucose dehydrogenase and
UDP-glucose-4-epimerase, both concerned with mucopolysaccharide synthesis,
were found to be elevated in 5-day-old can/can cartilage (Table 6). The nonspecific glucose-6-phosphate dehydrogenase was not elevated in can/can at
either 3 or 5 days.
Protein synthesis from [14C]glycine is significantly reduced in can/can cartilage
at birth and at 3 days (Table 7): by 5 days it is significantly higher than in
normal litter-mates, reflecting the increased mucopolysaccharide synthesis and
enzyme levels seen at this age. These changes in protein synthesis are not seen in
the liver.
Little of the protein synthesized by 3-day-old can/can mice is removed by
collagenase (Table 8): in 5-day-old mice both collagen and non-collagen
moieties are significantly increased.
If any of the abnormalities so far described were near the root cause of the
can achondroplasia it might be expected that they would be present before the
embryos can be classified externally. As a test of this, cartilage from 10 litters of
Biochemistry o/can cartilage
325
16
14
12
10
10
= 56
I
o
6
0L
6
4
0-5 10 1-5 20 2-5 30 3-5 40 4-5
dpm glucose incorporated,'
10 mg 2 hx 10 3
Fig. 1
8 10 12 14 16 18 20 22 24 26 28
dpm glycine incorporated/
10 mg/2 hx 10~3
Fig. 2
14
Fig. 1. Incorporation of [U- C]glucose in 17- and 18-day-old embryos from litters
segregating for can.
Fig. 2. Incorporation of [U-14C]glycine in 17- and 18-day-old embryos from litters
segregating for can.
embryos aged 17 and 18 days was incubated randomly with either [U-14C]glucose or [U-14C]glycine. The litters were derived from known + jean fathers
and mothers whose genotype was either +/can (P = f) or + / + (P = •§•).
Hence £ x •§• = $ of the embryos were expected to be can/can. The results presented as frequency distributions (Figs. 1, 2) show that the mucopolysaccharide
incorporation peak is unimodal, whilst that for protein synthesis is bimodal,
with ]-J (not significantly different from I) embryos comprising the lower peak.
The rates of incorporation into protein obtained also correspond well with
those seen in newborn canjean mice and normal litter-mates.
DISCUSSION
The earliest abnormality seen in can/can mice is a decrease in the rate of
protein synthesis in cartilage. This was seen in 17-day-old embryos which could
not be externally classified as abnormal. This decreased synthesis persists until
3 days post partum: at this time the amount of collagen being synthesized is
significantly lower than normal. The period between 3 and 5 days seems to be
326
D. R. JOHNSON AND D. M. HUNT
something of a landmark for the can/can mouse: protein synthesis (including
collagen synthesis) increases markedly, as does the incorporation of glucosamine into mucopolysaccharides and the levels of two mucopolysaccharide
synthetic enzymes. Perhaps all these represent a belated compensatory mechanism for previous inadequacies.
There are apparent anomalies in the data presented; the difference in total
glucosamine incorporation between normal and can I can is large at 5 days, but
incorporation into the protein-bound fraction is normal. One is tempted to
suggest that the synthetic mechanisms for the protein backbone(s) of hyaluronic
acid and chondroitin sulphate cannot keep pace with this abnormally high
synthesis, especially as the highest can/can rate (6-47 nmol/h at 3 days) is
similar to the highest normal rate seen (5-92 nmol/h at 5 days). This seems
unlikely, however, as the total rate of protein synthesis in canjcan cartilage is
high at this time.
In 3-day-old can/can mice, although total protein synthesis and collagen
synthesis are depressed (Table 8) collagenase-insoluble protein is increased. Is
this a portent of the 5-day situation with collagen synthesis lagging behind ?
The ratio of glucosamine: galactosamine incorporation is very high throughout. As chondroitin sulphate contains equal amounts of glucose and galactose
derivatives one would expect a priori something nearer to 1:1. Handley &
Phelps (1972) showed that the specific radioactivities of UDP-glucosamine and
UDP-galactosamine were identical after exposure to [U-14C]glucose, suggesting
rapid epimerization. However, glucosamine seems to be preferred, and Heyner
(1960) found that galactose failed to support the growth of foetal cartilage in the
absence of glucose.
The situation in can contrasts with that reported by Fraser & Goetinck (1971)
in the embryonic nannomelic chick. Here the amount of protein-bound 14C
label incorporated from glucosamine was significantly less than in normal
chicks; nannomelic chicks also incorporated 35S at a lower rate than normal. As
the chondroitin sulphate produced was of normal molecular weight, Fraser &
Goetinck conclude that the nannomelic chick produces fewer chains of chondroitin sulphate than its normal sibs.
In the achondroplastic (ac) rabbit utilization of glucose and galactose is
increased (Shepard & Bass, 1971). Bargman, Mackler & Shepard (1972) later
showed that the increased utilization of glucose compensates for a lack of
phosphorylation at the cytochrome oxidase region of the terminal oxidase
system of ac mitochondria. In canjcan the increased uptake of glucose is into
mucopolysaccharides: in addition the oxidative phosphorylation of cancan
liver mitochondria is normal (Table 9).
Seegmiller et al. (1971, 1972) described the ultrastructural appearance of
cartilage from the epiphyses and trachea of chondrodystrophic (cho) mice. They
observed a lack of metachromatic staining (common to all achondroplasias) and
an aggregation of collagen into banded fibres with a 640 A (64 nm) repeat.
Biochemistry o/can cartilage
327
Table 9. Oxidative phosphorylation in newborn + and can/can liver
+
can/can
2-179 ± 0-39 (4)
2-229 ± 0-40 (3)
Results are in /tmoles phosphorus esterified per mg liver/h±s.E.M.
Seegmiller suggests that the reduced mucopolysaccharide content implied by
lack of metachromia allows the tropocollagen to polymerize into banded' native'
collagen fibres.
Stanescu et al. (1972) looked at cartilage samples from seven distinct dwarfing
conditions in man: their main finding was a marked increase in collagen content
in the cartilage of a 4-5-day-old achondroplastic boy. This may be equivalent
to the post 5-day-old can Ican mouse which might be expected to show a fibrotic
matrix.
It is clear that the achondroplastic phenotype can be the end result of a
variety of very different first causes, and that can represents a type which differs
fundamentally from those previously described. It is also clear that our knowledge
of these first causes is minimal and that the achondroplastic phenotype presents
a field in which there is much scope for further research.
This work was supported in part by a grant from the Central Research Fund, University
of London. We thank Mrs P. Beveridge and Mrs S. Mansfield for technical help.
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