Amino acid and amino sugar in calcified tissues of portunid crab

Carey, F. G.

Degens, E. T.

Spencer, D. W.

English

NASA Technical Reports Server

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AMINO ACIDS AND AMINO SUGARS IN 
CALCI FI  ED TISSUES OF PORTUN I D CRABS 
WOODS HOLD OC?3MTOGiUEIIC INSTITUlON 
NSR-22-014-001 
by 
n (  
, Franc is  G. Carey, D r .  Derek W. Spencer and D r .  Egon T. Degens 
Woods Hole Oceanographic I n s t i t u t i o n ,  Woods Hole, Massachusetts. 
The va r ious  regions i n  t h e  exoskeleton of po r tun id  crabs e x h i b i t  a wide range 
of hardness and r i g i d i t y .  The most r i g i d  s t r u c t u r e  i s  t h e  dac ty lus  of t h e  chela; 
most f l e x i b l e  and s o f t  are t h e  unmineralized j o i n t s  between t h e  lirnbs. The pero- 
podus, t h e  carapace and The pleopods represent in termediate stages o f  r i g i d i t y .  
Since t h e  ex ten t  o f  m i n e r a l i z a t i o n  la rge ly  determines t h e  f l e x i b i l i t y  and hardness 
o f  t h e  exoskeleton, we were i n te res ted  i n  learn ing  t h e  important f a c t o r s  i n  t h i s  
c a l c i f i c a t i o n  process. 
Prev ious work on m i nera I i z a t  i on i n  b i o I og i ca I systems ' has shown t h a t  t h e  
proteinaceous m a t r i x  i n  c a l c i f i e d  t i ssues  prov ides a s e t  o f  h i g h l y  s p e c i f i c  tem- 
, p la tes .  Most essen t ia l  i n  nuc lea t ing  a minera l  phase appears t o  be t h e  a v a i l -  
a b i l i t y  of f ree  carboxyl and amino groups prov ided by c e r t a i n  a c i d i c  and bas ic  
amino ac ids.  I n  t h e  l i g h t  of these r e s u l t s  we decided t o  determine t h e  amino 
a c i d  and amino sugar composition o f  representa t ive  reg ions i n  t h e  exoskeleton 
and t o  r e l a t e  these d a t a  t o  t h e  c a l c i f i c a t i o n  phenomena. 
The animals se lected f o r  t h i s  study were f o u r  specimens o f  CalZizzectes 
sapidus, one o f  OvaZipes oceZZatus, and one of Careinides mamas, a !  I i n t e r m o l t  
males. 
t h e  carpus and merus o f  t h e  che!iped, t h e  f l e x i b l e  p s i d l g  bf  t h e  pleopod, t h e  
ca rd iac  and g a s t r i c  regions -.f t h e  carapace, t h e  propodus and dac ty lus  o f  t h e  
The regions sampled were t h e  s o f t  u n c a l c i f i e d  j o i n t  membrane between 
https://ntrs.nasa.gov/search.jsp?R=19670018378 2020-03-12T11:36:18+00:00Z
I 
. .  . 
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, -  
chel iped.  
c i f i c a t i o n ,  hydro lys is ,  and ion-exchange chromatography . 
nes i um and s t  ron t  i urn obta ined by atom i c absorpt ion spectroscopy3 and on phosphate 
by c o l o r i m e t r y  
A l l  samples were freed o f  extraneous t i s s u e s  and subjected t o  decal- 
2 Data on calcium, mag- 
4 
were used as a measure of t h e  degree o f  c a l c i f i c a t i o n  o f  t h e  i n d i -  
v idua l  o rgan ic  mat r ix .  The a n a l y t i c a l  r e s u l t s  a re  
I n  o rder  t o  exp lo re  t h e  i n t e r r e l a t i o n s h i p s  both w i  
tween t h e  amino compounds and CaCO t h e  technique 
employed . However, as our  calcium data are  r e s t r  
3' 
5 
summarized i n  Tab les I and 2. 
h i n  t h e  amino ac ids and be- 
of f a c t o r  ana lys i s  has been 
c t e d  t o  t h e  sample o f  Ca l l i nec -  
tes, we conf ined o u r  f a c t o r  ana lys i s  on ly  t o  t h e  c a l c i f i e d  t i s s u e s  of these 
specimens. 
amino sugar t o  p r o t e i n  ra t i o ,  and t h e  calcium content  i n  a l l  samples. The anaiy- 
s i s  was performed by a GE 225 computer using a program w r i t t e n  by Spencer . A 
p r i n c i p a l  components s o l u t i o n  f o l  lowed by a varimax r o t a t i o n  -showed t h a t  t h r e e  
f a c t o r s  account f o r  83% o f  t h e  variance o f  t h e  data. The varimax f a c t o r  m a t r i x  
and t h e  varimax f a c t o r  score m a t r i x  are reproduced i n  Tables 3 and 4. F igure I 
i s  a p l o t  of t h e  f a c t o r  scores of  f a c t o r  I and f a c t o r  2. 
I n  a l l ,  we analyzed t h e  r e l a t i o n s h i p s  between I S  amino acids, t h e  
6 
About 80% by weight o f  t h e  samples when expressed as carbonates, amino acids, 
and acetylglucosamine polymer could be accounted f o r  a f t e r  t h e  chemical ana lys is .  
The miss ing  p o r t i o n  p a r t l y  represents humin and p a r t l y  water; c h i t i n  i s  known t o  
r e t a i n  up t o  10% water even when d r i e d  t o  constant  weight a t  105'C7. 
Based on t h e  f a c t o r  analysis, t h e  f o l  lowing i n t e r p r e t a t i o n s  are o f fe red .  
Factor  one i s .  r e l a t e d  t o  t h e  ac tua l  c a l c i f i c a t i o n  process. I n  t h i s  fac to r ,  pro- 
l ine ,  lys ine,  and t h e  amino sugar t o  p r o t e i n  r a t i o ,  form a cova r ian t  group s t rong-  
l y  c o r r e l a t e d  w i t h  the  ca lc ium content  and nega t i ve l y  c o r r e l a t e d  w i t h  a s p a r t i c  
acid, threonine,  serine, g lyc ine ,  t y ros ine  and phenylalanine. Fac tor  two forms 
a cova r ian t  group i n v o l v i n g  iso leuc ine,  leucine, va l ine,  g lu tamic  a c i d  and a la -  
-3- 
nine.  Fac tor  two forms a covar ian t  group invo lv ing  iso leuc ine,  leucine, va l ine,  
g lu tamic  a c i d  and alanine. Noteworthy i s  t h e  f a c t  t h a t  ind i 'v idual  number 12 
(Table 4 )  general l y  scores h igher  on f a c t o r  two than e i t h e r  i n d i v i d u a l  number I !  
or  13, as i l l u s t r a t e d  i n  F igure 2. Although t h e  pleopod, propodus and carapace 
of  these i n d i v i d u a l s  conta in  l a r g e r  q u a n t i t i e s  o f  t h e  amino ac ids invo lved i n  
t h i s  fac to r ,  f a c t o r  two appears t o  have no connection t o  t h e  actual  c a l c i f i c a -  
t i o n  process. Because of t h e  d i f fe rences  i n  i n d i v i d u a l s  it seems most l i k e l y  
t o  be an environmental f a c t o r  (e.g. water temperature, pH, Eh, s a l i n i t y ,  o r  
d i e t ) .  Factor  3 has a loading on bas ic  amino ac ids.  I n  comparison w i t h  f a c t o r s  
I and 2, it con t r i bu tes  I itt l e  t o  the  f a c t o r  score. 
The increase i n  l y s i n e  and OH-lysine w i t h  progressing c a l c i f i c a t i o n  aarees 
w i t h  t h e  concept'  t h a t  bo th  amino acids may prov ide nuc lea t ion  s i t e s  f o r  c r y s t a l  
growth. 
y i e l d s  of glucosamine i n  t h e  most minera l i zed  reg ions o f  t h e  exoskeleton may a l s o  
be l i nked  t o  c a l c i f i c a t i o n .  Should d i ca rboxy l i c  ac ids be essen t ia l  i n  p r o v i d i n g  
negat ive  s i t e s  f o r  t h e  f i x a t i o n  o f  calcium, t h e i r  e f f e c t  i s  masked by o the r  fac- 
t o r s .  
Inasmuch as c h i t i n  may a l s o  conta in  f ree  amino g r o ~ p s ~ - ~ ,  t h e  h igher  
Crustacean c u t i c l e s  are hardened by bo th  tanning and m i n e r a l i z a t i o n  processes. 
As a general r u l e  on increase i n  tanning i s  accompanied by a reduc t ion  i n  minera!  
depos i t ion  and v i ce  versa. Thus a t  least two d i s t i n c t  p r o t e i n  ma t r i ces  are  con- 
t a i n e d  i n  t h e  r i g i d  s t r u c t u r e s  o f  t h e  exoskeletcn. Th is  phenomenon i s  analogous 
t o  t h e  occurrence o f  minera l i zed  pro te ins  and t h e  per iost racum i n  mo l lusk  s h e l l s .  
A study o f  t h e  amino ac id  composition i n  tanned p r o t e i n s  o f  gastropods and cep- 
halopods i s  in fo rmat ive  f o r  t h e  i n t e r p r e t a t i o n  o f  t h e  cova r ian t  group i n  fac+or 
one, i.e. a s p a r t i c  acid, threonine, serine, g lyc ine ,  t r y o s i n e  and phenyla lan ine.  
E s s e n t i a l l y  t h e  same amino ac ids charac ter ize  t h e  per iost racum o f  these two 
c lasses e f  mol!uscs . This  s i m i l a r i t y  i s  f u r t h e r  under l ined  by t h e  h igh  ahund- 9 
-4- 
ance o f  amino sugars i n  t h e  tanned s h e l l  p ro te ins .  I n  t h e  l i g h t  o f  these data, 
f a c t o r  one (Table 3) can be regarded as a r e f l e c t i o n  o f  t h e  d i f f e r e n t  mix ing ra- 
t i o s  o f  m ine ra l i zed  and tanned p r o t e i n s  i n  crustacean c u t i c l e s .  The assoc ia t ion  
of a s p a r t i c  ac id  w i t h  t h e  tanned p ro te ins  and o f  p r o l i n e  w i t h  t h e  minera l i zed  
t i s s u e s  does no t  necessar i l y  imply t h a t  the  former i s  no t  invo lved i n  t h e  ac tua l  
c a l c i f i c a t i o n  process whereas t h e  l a t t e r  i s .  
unre la ted  t o  c a l c i f i c a t i o n  t h e  minera l i zed  t i s s u e s  conta in  less a s p a r t i c  a c i d  
and more p r o l i n e  than t h e i r  tanned counterparts. 
m i n e r a l i z a t i o n  vary from region t o  region as shown by representa t ive  values f o r  
t h e  th ickness  o f  tanned and minera l i zed  layers i n  CaZinectes presented i n  Table 
5. The s t rong  negat ive c o r r e l a t i o n  between t h e  two groups of amino ac ids i n  fac- 
t o r  one i s  probably r e l a t e d  t o  t h e  changing r a t i o  o f  tanned t o  minera l i zed  
I t  may be t h a t  because o f  processes 
The p ropor t i ons  o f  tanning and 
c u t i c l e  i n  t h e  var ious  regions. The other  two specimens inc luded i n  t h i s  repor t ,  
namely, OvaZipes oceZZatus and Careinus maenas show e s s e n t i a l l y  t h e  same f a c t o r  
s t r u c t u r e  and biochemical r e l a t i o n s h i p s  we discussed before.  The enrichment i n  
l y s i n e  and amino sugars w i t h  progress ive c a l c i f i c a t i o n  i s  even more pronounced 
than i n  Callinectes and may be a species c h a r a c t e r i s t i c .  
Based on t h e  amino ac id  composition, t h e  j o i n t  membrane d i f f e r s  i n  sane as- 
pects  from t h e  minera l i zed  s t r u c t u r e s  so f a r  considered. For t h i s  reason, we 
excluded t h e  data i n  ou r  f a c t o r  program. P a r t i c u l a r l y  no eworthy i s  t h e  h igh  
abundance of p ro l i ne ,  ac id ic ,  aromatic and bas ic  amino ac ds. 
I n  r e l a t i n g  t h e  present data on minera l i zed  t i s s u e s  n p o r t u n i d  crabs w i t h  
prev ious r e s u l t s  l a r g e l y  i n fe r red  from electronmicrographs" we o f f e r  a ten ta -  
t i v e  model on t h e  c a l c i f i c a t i o n  i n  t h i s  b i o l o g i c a l  system: The c u t i c l e  o f  crus-  
tacea i s  a layered s t r u c t u r e  composed of m ine ra l s  deposited i n  and between matted 
layers  of c h i t i n  and p r o t e i n  f i b r i l s .  The f i b r i l s  ma in ly  l i e  i n  t h e  plane of t h e  
-5- 
layers, b u t  some branch up and down t o  connect adjacent layers.  The v e r t  ca t  f i b -  
r i l s  l i n e  t h e  w a l l s  o f  t h e  numerous pore canals  which run through t h e  c u t  c l e  i n  a 
d i r e c t i o n  perpendicu lar  t o  i t s  surface. 
between t h e  f i b r i l s  and i n  t h e  pores. 
bu t  t h e  r e s u l t i n g  c r y s t a l s  are always small i n  s ize .  
ed t o  t h e  i n t e r s t i c e s  and lumen o f  t h e  pore canals. I t  i s  i n f e r r e d  t h a t  t h e  form; 
a t i o n  of mu l t i t udes  of small minera l  seeds i n  t h e  f i b e r s  represent t h e  f i r s t  
c r y s t a l l i z a t i o n  stage. The depos i t ion  of l a rge  minera ls  comes l a t e r  as nuclea- 
t i o n  s i t e s  i n  t h e  m a t r i x  become gradua l ly  avai table. The la rge r  s i z e  of t h e  
c r y s t a l s  i n  t h e  second m i n e r a l i z a t i o n  phase i s  simply a consequence o f  t h e  fewer 
nuc lea t i on  s i t e s  a v a i l a b l e  along t h e  organic  templates. The i n d i v i d u a l  layers 
a re  spaced f u r t h e r  apar t  i n  t h e  th i ck ,  h i g h l y  c a l c i f i e d  reg ions compared t o  t h e  
less minera l i zed  regions. The proteinaceous ma t te r  i s  p r i n c i p a l l y  contained i n  
t h e  layers  o f  matted f i be rs ,  and consequently, t h e  most c a l c i f i e d  reg ions have 
a g r e a t e r  p ropor t i on  o f  minera l  t o  organic mat te r .  Th i s  imp l ies  t h a t  w i t h  ad- 
vancement of c a l c i f i c a t i o n  t h e  organ ic  mat r ix  becomes a more e f f e c t i v e  template. 
I n  a sense t h i s  i s  a d u p l i c a t i o n  of what we observe i n  mol lusk s h e l l s  by going 
9 from p r i m i t i v e  t o  h i g h l y  advanced forms , 
organ ic  m a t r i x  i s  requ i red  for t h e  nuc lea t ion  of ca lc ium carbonate. Whereas a 
NautiZus, HaZiotis, o r  MytiZus may requi re a few pe r  cent  o rgan ic  ma t te r  f o r  t h e  
depos i t ion  of t h e i r  s h e l l  s t ruc tu re ,  sane h i g h l y  evolved gastropods such as 
Archi tectmica o r  BuZZa 
Organic m a t e r i a l s  form a d i f f u s e  m a t r i x  
M i n e r a l i z a t i o n  proceeds along t h e  f i b r i l s  
Larger c r y s t a l s  a re  r e s t r i c t -  
-~ 
With evoIut ion,progressiveIy less 
g e t  along w i t h  j u s t  0.01%. 
-6- 
. REFERENCES 
. I .  F lo rk in ,  M., Gregoire, C., Bricteux-Gregoire, S., and Schof fen ie ls ,  E., 
.C.R. Acad. Sci., Paris; 252, 440 (1961). 
Glimcher, M.J., i n  C a l c i f i c a t i o n  i n  B i o l o g i c a l  Systems, 421 ( P u b l i c a t i o n  
No. 64 of  t h e  American Associat ion f o r  t h e  Advancement o f  Science, 
1960). 
Gregoire, C., Bu l l . ,  I ns t .  Roy. Sci. Nat. Belg., 35, I (1959). 
Hare, P.E., Ph.D. thes is ,  C a l i f o r n i a  I n s t i t u t e  o f  Technology (1962); .Science, 
139, 216 (1963). 
Moss, M.L., ed., Comparative Biology of C a l c i f i e d  Tissue: Ann. N.Y. Acad. 
Sci., 109 (1963). C a l c i f i c a t i o n  i n  B i o l o g i c a l  Systmes, e d i t .  by 
Sognnaes, R.F. (Pub l i ca t i on  No. 64 o f  t h e  American Associat ion 
for  t h e  Advancement o f  Science, 1960). Mechanism o f  Hard T issue 
Destruct ion,  e d i t .  by Sognnaes, R.F. ( P u b l i c a t i o n  No. 75 o f  t h e  
American Associat ion for  the  Advancement o f  Science, 1963). 
Wilbur, K.M., i n  C a l c i f i c a t i o n  i n  B i o l o g i c a l  Systems, 15 ( P u b l i c a t i o n  No. 
64 o f  t h e  American Associat ion for  t h e  Advancement o f  Science, 
1960). 
2 .  Degens, E.T. and Spencer, D.W., Technical Report Woods Hole Oceanographic 
I n s t i t u t i o n ,  Reference No. 66-27. (1966). 
3. The Perk in  Elmer Corp., Ana ly t i ca l  Methods for  Atomic Absorption Spectro- 
photometry (1966). 
4. A I  len, R.J.L., Biochem. J., - 34, 858 (1940). 
i c 
3 .  iiannan, S.H., Modern Facior  Anaiysis, u n i v e r s i i y  o i  Lnicago Fress, i%o. 
6, Spencer, D.W., Factor  Analysis. Technical Report, Woods Hole Oceanographic 
I n s t i t u t i o n ,  Ref. No. 66-39. (1966). 
7. Bai ley,  K. and Weiss-Fogh T.., Biochim. Biophys. Acta, - 48, 452 (1961). 
a. Hackman, R.H. Aust. J .  b i o l .  Sci., - 13, 568 (1960). 
9. Degens, E.T., Spencer, D.W. and Parker R.H., Comp. Biochem. Phys io l . ,  - 20, 
553 (1967). 
IO. 
1 1 .  This  work was sponsored by g ran ts  No. 22-001-014 from t h e  Nat ional  Aero- 
Trav is ,  D.F., Ann. New York Acad. Sci., - 109, 177 (1963). 
nau t i cs  8, Space Admin is t ra t ion and No. 816 from t h e  Nat ional  
Science Foundat ion. 
-
0 * 
Table 2 
.. Element Composition i n .  Various Regions of t h e  Exoskeleton of CaZZinectes 
Weight % J o i n t  Membrane P I eopod Carapace 
Ca I ci urn 0.05 13.5 23.4 
Magnes i urn 0.03 0.87 I .01 
S t  r o n t  i urn - 0.27 0.23 
Phosphorus I .7 0.9 
Propod us Dacty I us 
25.2 27.7 
1.19 I .26 
0.22 0.26 
I .3 I .2 
Table 3 
Va r i max Factor M a t r i x  
Aspar t i c  Acid 
Th reon i ne 
Ser ine 
Glutamic Acid 
Pro1 i ne 
G I  y c i  ne 
A I  an i  ne 
Cyst ine 
Val ine 
Methionine 
Iso leuc ine  
Leuci ne 
Tyros i ne 
Phenylalanine 
Hydroxy I ys i ne 
Lys i ne 
H i  s t  i d i ne 
Arg in ine  
Amino Sugar/Protein 
Ca I c i  um 
I 
0.923 
0.751 
0.927 
-0.736 
0.825 
-0.668 
-0.599 
0.926 
0.897 
-0.847 
-0.468 
-0.831 
-0.926 
Factor  
2 3 
0.819 
-0.560 
0.729 
0.919 
0.401 
0.877 
0.730 
0.91 I 
0.842 
-0.344 0.896 
Table 4 
Varirnax Factor Score M a t r i x  
Sample No. 
13 
12 
I 1  
12 
I 1  
13 
12 
1 1  
13 
12 
I 1  
Region o f  
Exoske I e ton 
PI  eopod 
P I eopod 
P I eopod 
P ropod us 
P ropod us 
Propodus 
Carapace 
Carapace 
Carapace 
Dacty I us 
Dacty I us 
I 
I .320 
I .299 
0.999 
0.022 
-0.948 
-0,766 
-0.125 
0.593 
0.171 
-I .730 
-0.835 
2 
-0.956 
-0.080 
- I  .408 
0.703 
-0.534 
-0.793 
I .778 
I .416 
0.371 
-0.610 
0. I13 
3 
1,456 
- I  .243 
-0.777 
I .657 
-0.488 
-0.180 
-0.695 
-0.493 
0.121 
'-0.619 
I .261 
c 
L 
Table 5 
Th ickness of  Tanned and M i  nera I ized Reg ions i n CalZinectes 
, 
P I eopod 
Carapace 
Propodus (che I a )  
Dactylus (che la )  
E p i c u t i c l e  and Pigmented Layer Endocut ic le  -
(mm) (mm) 
0.06 
0.06 
0.06 
0.07 
0.25 
0.45 
0.75 
I .30 
The e p i c u t i c l e  and pigmented layers are tanned and c a l c i f i e d .  
The Endocut ic le  i s  c a l c i f i e d .  
THE LOADING OF FACTOR I VERSUS F A C T O R I T  
1.0 
FACTOR I 
T Y R  SEf? 
PHE 
.8 
T H R  
.6 
.4 
.2 
0.8 0.6 - .4 - .2 
c I I I I I I ' A R ~  CY 5 
PRO 
0.2 
- .4 
HIS 
- .6 
- .8 LY s 
-1.0 
ASP 
; LY 
I so 
FACTOR IT 
.2 .4 .6 .8 
OHMLYS 
MET 
A M W P  
CA 
G LU 
VA L 
ALA 
LEU 
FIG.1 
FACTOR 11: SCORES IN 3 INDIVIDUALS (11,12 AND 13) 
OF CALLINECTES SAPIDUS. 
FIG. 2 


Amino acids and amino sugars in calcified tissues of portunid crabs

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