The amounts of nonmineralized and mineralized collagen in bone from control, immobilized, and immobilized reambulated monkeys were examined. In order to understand structure function relationships of bone collagen and the reponse of a variety of conditions on control of the three dimensional structure of the collagen fibril, the stereochemistry of the cross-linking reactions as well as the stereospecific packing of the collagen molecules were studied

Mechanic, Gerald L.

English

NASA Technical Reports Server

kechnical Report 
January 1, 1986-December 31, 1986 
NASA Grant NAG 2-181 
Principal Investigator: Gerald L. Mechanic, Ph. D. 
Title: Biochemical Changes in Bone in a Model of 
Weightlessness 
The paper concerned with the amounts of 
nonmineralized and mineralized collagen in bone from, 
control, immobliized, and immoblized reambulated 9 
m 
W 
In 
I * 
W 
F 
(various periods of time) monkeys was published. The 
scientific response to the paper has been.positive and 
many requests have been made for reprints. 
In order to understand structure function 
d 
a 
relationships of bone collagen and the responses of a u 
variety of conditions on control of the three 
N 
tn 
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a: rJ2 5 
been studying the stereochemistry of the cross-linking ;r; u4 
reactions as well as the stereospecific packing of the 
collagen molecules. We have chosen, as models, s k i n ,  
periodontal ligament since the former has a different 
form of stable cross-link and the latter does not 
contain stable cross-links. However, the latter upon 
incubation in vitro forms pyridinoline, the same cross- 
link that exists in bone collagen. A s  a model for the 
phosphoprotein interaction with hard tissue mineralized 
collagen we have chosen dentin because of its relative 
https://ntrs.nasa.gov/search.jsp?R=19870006250 2020-03-20T12:07:05+00:00Z
G.L. Mechanic Tech. Rep. NAG 2-181(contad) 
simplicity when compared with bone. The former does 
not turn over while the latter is always remodeling. 
Investigations of this sort will allow us to explain 
the reactions taking place on a molecular and 
biochemical level. By knowing the latter we shall 
derive valuable information on the mineralization of 
bone, where the crystallites reside and their size. 
Progress along these lines are summarized below. 
In 1982 we isolated as an apparently homogeneous 
peptide, a three-chained peptide containing the stable 
non-reducible trifunctional cross-link, which w a s  then 
known at the time as hydroxyaldolhistidine (HAH) (13). 
Based on the structure proposed by Housley et al. (64) 
we attempted to specifically cleave the peptide by 
cleaving the cross-link’s putative C-C enamine double 
bond with OsO4/NalO4. From the reaction mixture we 
obtained a two-chained peptide whose structure after 
amino acid and sequence analysis was deduced as alCB4- 
5(76-90) [Hyl-873 X alCB6(S -18c 1 CLy~3-16~ 3 .  
Designations of cross-linked peptides used here and in 
the remainder of the proposal are based on the CNBr 
peptides derived from collagen; the numbers in 
parentheses represent the residue number6 in the 
sequence; the residue and its number that are in 
brackets represents the cross-linking residue in the 
peptide; and the X denotes the cross-linking moieties. 
The structure and the identity of the third chain 
eluded us as it did Becker et al., (1975). Recently we 
have identified the third chain as a2CB4(76-126)CHis- 
921. The structures of the tryptic peptides isolated 
were alCB4-5(76-90)IHyl-873 X alCB6(993-22C)[Lys-16c1 X 
a2CB4(76-126)CHis-923 and alCB4-5(76-90)CHyl-873 X 
alCB6(993-22= 1 C L ~ s - 1 6 ~  1 X a2CB4(91-126) [His-923. The 
peptides isolated from the latter two peptides after 
treatment with bacterial collagenase were alCB4-5(85- 
90)CHyl-873 X alCB6(1012-22C ) C L y ~ - 1 6 ~ 3  X a2CB4(85- 
99)CHis-923 and alCB4-5(85-90)CHy1-873 X alCB6(1012- 
2 2 C ) C L y ~ - 1 6 ~ 3  X a2CB4(91-99)CHis-923. Measurements 
from the C-A of His to the C-A of Lys obtained from a 
space filling model of the cross-link and modeling 
performed by Dr. Elton P. Katz strongly suggested that 
the trifunctional cross-link linked three molecules of 
collagen together in the fibril. The data also 
suggested that skin collagen molecules are packed in 
fibrils differently from those of periodontal ligament. 
In the course of other work we noted a precursor- 
2 
G. L. Mechanic Tech. Rep. NAG 2-181 (cont"d) 
product relationship between dehydro-hydroxylysino- 
norleucine and this trifunctional cross-link. We 
therefore prepared a large amount of the cross-link and 
subjected it to Fast Atom Bombardment Mass 
Spectrometry, 'H- and I a C -  Nuclear Magnetic Resonance. 
The structure found was consistent with 
histidinohydroxylysinonorleucine (HHL). Studies of the 
concentration of the cross-link in human and bovine 
skin collagen indicated it constantly increased with 
chronological age of the organism, approaching one 
residue per mole of collagen (0.9 residues per mole 
collagen in 82 year-old human skin). This is quite 
different from the trifunctional stable cross-link, 
pyridinoline, which increases during the growth period 
of the organism and then decreases in abundance with 
aging. This cross-link,HHL, is the first demonstration 
of a collagen cross-link that shows a continuous 
increase with chronological age of organism. Based on 
glycosylation data it was proposed that the function 
of specific glycosylated Hyl residues was to prevent 
cross-links in tissue collagen from maturing to stable 
cross-links so tissue turnover could occur more 
readily. This vas borne out by our studies on the 
periodontal ligament which is the fastest turning over 
collagen in the body. 
A trypsin digest of denatured NaBJ H. -reduced 
native bovine periodontal ligament was prepared and 
fractionated by gel filtration and cellulose ion- 
exchange column chromatography. Prior to trypsin 
digestion a complete acid hydrolysate was subjected to 
analyses for non-reducible stable and reducible 
intermolecular cross-links. Minute amounts of the 
former and significant amounts of the reduced cross- 
links, dihydroxy-lysinonorleucine (1.1 moles/mole 
collagen), hydroxylysinonorleucine (0.9 mole/mole 
collagen) and histidinohydroxymerodesmosine (0.6 
mole/mole collagen) were found. The covalent 
intermolecular cross-linked two-chained peptides that 
were isolated were subjected to amino acid and sequence 
analyses. The analyses indicated that the structures 
for the different two-chained linked peptides were 
alCB4-5(76-90)CHyl-B71 X alCB6(993-22C ) C L y ~ ~ ~ ~ - 1 6 ~ 3 ,  
alCB4-5(76-90)CHyl-873 X alCB6(993-22c)CHyla1d-16C3, 
a2CB4(76-90) CHyl-873 X alCB6(993-22C ) CLysal -16c 3, and 
a2CB4(76-90) CHyl-871 X alCB6(993-22C ) [Hyla' -16c 1. All 
the cross-link peptides isolated were solely from Type 
I collagen. This is the first direct evidence to 
demonstrate involvement of an a2 chain in the cross- 
linking of collagen. Alkaline hydrolysis indicated 
that all the cross-links in the peptides were 
completely glycosylated. Quantitative determination of 
the amounts of the various two-chained peptides present 
in the collagen coupled with their molecular locations 
3 
G. L. Mechanic Tech. Rep. NAG 2-181 (cont"d) 
indicated a stoichiometric conversion of residue 16c 
aldehyde to intermolecular cross-link in each of the 
COOH-terminal non-helical peptide regions of both a1 
chains in a molecule of Type I collagen. Analyses from 
our data of the ratio of a1 to a 2  intermolecularly 
cross-linked chains involved at the latter locus 
(residue 16c ) showed it to be 3 . 3 : l .  This strongly 
indicates that the three dimensional molecular packing 
of Type I collagen molecules on a hexagonal lattice in 
bovine periodontal ligament is not random but 
stereospecific in nature and also indicates that the 
molecules are in a specific azimuthal (angular) 
orientation with each other. This is the first time 
chemical evidence has been obtained to indicate the 
conclusions concerning the three dimensional packing of 
collagen molecules in fibrils. This paper was 
published in Biochemistry this year. In addition we 
found the cross-links completely in their glycosylated 
form (Table 3 of the paper). We proposed as above that 
gycosylation of specific Hyl residues might be 
necessary to prevent stable non-reducible mature cross- 
link formation for virtually no stable non-reducible 
mature cross-links were found in the periodontal 
ligament collagen. 
Embryonic bovine dentin (8-8.5 month-old embryo) 
was separated into reduced-nonmineralized fraction 
(Red-NMF), mineralized fraction-EDTA extract (MF-ED) 
and reduced-mineralized fraction (Red-MF) portions as 
described in the methods below. The yield (weight) was 
approximately 1 to 0.1 to 1.1 for each fraction 
respectively. This ratio is significantly different 
from normal bovine bone in which case the ratio was 1 
to 0.1 to 3 (7 month-old embryo) and 1 to 0.1 to 4 ( 2  
year-old bovine bone). Cross-link analyses indicated 
the major reduced cross-link was dihydroxylysino- 
norleucine in each case. Quantification has not been 
accomplished as yet. 
The collagen content in each fraction was 
approximately 60% in Red-NMF, 10% in MF-ED and 95 % in 
Red-MF based on a value of 300 residues of Hyp per 
collagen molecule. 
Digestion of MF-ED and Red-MF with trypsin almost 
completely solubilized each of these fractions. Equal 
collagen aliquots (based on Hyp analysis) of the 
tryptic digests of Red-NMF (see above), Red-MF and 1/5 
the amount of MF-ED by weight (limited quantities of 
this fraction are obtained and very small amount of 
collagen was present) were each subjected to molecular 
sieve column chromatography using Sephadex G50 SF (see 
methods and ref 9 in appendix). The MF-ED profile 
contained one large peak in close to the void volume of 
4 
G.L. Mechanic Tech. Rep. NAG 2-181 tcont’d) 
the column (>20,00O‘dalton). No radioactivity nor 
fluorescence were detected. In the case of Red-NMF 
and Red-MF, the most prominent radioactive peak 
appeared in the region of the standard cross-linked 
peptide alCB6(993-22C )CHyl-16c3 X alCB4-5(76-90)CHyl- 
873 obtained f r o m  bovine periodontal ligament. This 
fraction was designated as peak V I 1  in our recent 
report. The minor radioactive peak was observed in the 
position where the standard cross-linked peptide alCB1- 
2(lN-9)CLys-9”3 X alCB6(928-933)CHyl-9303 eluted from 
the column. This fraction was designated as peak X in 
the same report. Three distinct flourescent peptide 
peaks were also observed in both profiles. The most 
prominent flourescent peak in each case eluted at the 
same position as alCB6(993-22c )IHyl-16c3 X alCB6(993- 
22c )CHyl-16c3 X alCB4-5(76-90)CHyl-673 which we have 
isolated from bovine tendon and bone collagen as well 
as 3 months old osteogenic osteoblastoma chick bone 
collagen and have previously isolated from dentin. The 
second flourescent peak in the profile eluted in the 
approximate molecular weight range of 8000 daltons and 
its structure is as yet unknown. The third flourescent 
peak contains a leading edge which contains a 
flourescent peptide whose structure is still not known 
while the main peak contains the cross-linked peptide 
alCB1-2(1” -9) CHyl-9” 3 X alCB1-2(lN -9) CHyl-9” 3 X 
alCB6(928-933)CHy1-9303 which we have also isolated 
from bovine tendon. Although the overall elution 
patterns of radioactivity and fluorescence showed some 
similarity between Red-NMF and Red-MF, the ratio of the 
major fluorescent peak to the major radioactive peak is 
less in Red-MF than in Red-NMF, indicating 
significantly more pyridinoline cross-link containing 
peptides in NMF than in MF. 
In our examination of the nonmineralized and 
mineralized collagen in bone we have noted that a small 
amount of phosphoprotein is bound covalently to 
collagen o n l y  in the mineralized fraction. Similar 
studies using dentin fractions described above as a 
model were performed. Organic phosphorus ( P )  assays 
were carried out on each fraction obtained from the 
Sephadex GSOSF gel filtration column. The major P 
containing peak from Red-NMF eluted in the 3,000 dalton 
range while those from MF-ED and Red-MF appeared in the 
5 
G.L. Mechanic Tech. Rep. NAG 2-18l(contad) 
high molecular weight range from the column (>20,000 
dalton). DEAE-cellulose chromotograpy of the P 
containing peak from Red-NMF provided 5 sharp peaks 
with only the 4th containing P which eluted at 0.15 M 
N a C 1 .  Amino acid analysis indicated it was 1/4 Pro. 
This could represent a fragment of a proteoglycan since 
its amino acid composition was not characteristic of 
phosphoprotein. It is known that proteoglycan is 
present in predentin. The P containing fraction from 
MF-ED separated into three peaks on the same column. 
The first peak was broad and amino acid analysis 
indicated it was somewhat collagenous in nature, it 
contained no P. The second peak eluted at 0.4 M NaC1. 
It contained a large amount of P and it’s amino acid 
composition indicated that 85% of the amino acids were 
A s p  and Ser. The amino acid profile also contained a 
peak at the elution position of phospho-Ser. The 
latter three characteristics are typical of dentin 
phosphoprotein. The third peak eluted at 0.55 M NaCl 
and also contained P. Its amino acid composition was 
non-descript. The P containing fraction from the GSOSF 
column derived from Red-MF contained two approximately 
equal sized peaks in the DEAE-cellulose elution 
profile. The first was broad and heterogeneous eluting 
between 0.05 and 0.15 M NaCl and did not contain P, 
however, the second peak eluted as a single symmetrical 
peak at 0.4 M NaC1. Amino acid analysis demonstrated 
6 
G . L .  Mechanic Tech. Rep. NAG 2-18l(contWd) 
that 80% of the total amino acids consisted of Asp and 
Ser. In addition a peak typical of Ser phosphate w a s  
present at the void volume of the amino acid profile. 
The latter two chacteristics are typical of a 
phosphoprotein. This DEAE-cellulose fraction also 
contained significant amounts of Hyp and Hyl. 
Additionally there was present a significant quantity 
of histidinoalanine. The latter is absent from the P 
containing fraction from Red-NMF. These results 
indicate that only the P containing fraction from the 
mineralized portion of the dentin contains the 
histidinoalanine, a cross-link that has been suggested 
to be associated with dentin phosphoprotein previously. 
The fact that Hyp and Hyl were also present in the same 
protein peak could very well mean that the collagen 
portion is covalently linked to the phosphoprotein via 
the histidinoalanine present. Phosphoprotein linked to 
collagen peptides have been proposed previously. 
Further work will be needed to demonstrate whether or 
not this protein peak is homogeneous and to 
characterize this peptide. Its structure will be 
identified in future work. 
In addition we have analysed bones of rats that 
were aboard Shuttle Spacelab 3 in collaboration with 
Dr. Sara Arnaud. W e  found that the concentration of 
collagen in the bone was increased. The results are 
now in press in the American Journal of Physiology. 
7 
G. L. Mechanic Tech. Rep. NAG 2-181 (cont"d1 
Papers published this year that w e r e  sponsored in 
part by N A S A  Grant NAG 2-181 and that were stimulated 
by the work to be performed for the research program 
.- 
are : 
Mechanic, G . L . ,  Young, D.R., Banes, A . J . ,  and 
Yamauchi, M. "Non-Mineralized and Mineralized Bone 
Collagen in Bone of Immobilized Monkeys" Calcif. 
Tissue Int. 39, 63-68 (1986) 
Patterson-Buckendahl, P., Arnaud, S . ,  Mechanic, 
G . L . ,  Martin, R.B., Grindeland, R.E., & Cann, C.E. 
"Bone Fragility and Composition of Growing Rats 
After One Week in Spaceflight" Amer. J. Physiol. 
In Press (1986) 
Graham, L. & Mechanic, G. L. " CJAcrylonitrile: 
Preparation Vie a Stable Tosylate Intermediate and 
Quantitative Reaction With Amine Residues in 
Collagen" Anal. Biochem. 153, 354-358 (1986) 
Yamauchi, M., Katz, E.P., & Mechanic, G. L. 
"Intermolecular Cross-linking and Stereospecific 
Molecular Packing in Type I Collagen Fibrils of 
the Periodontal Ligament" Biochemistry 25. 
4907-4913 (1986) 
Mechanic, G.L., Katz, E.P., Henmi, M., Noyes, C. & 
Yamauchi, H. "Locus of a Histidine-Based, Stable, 
Trifunctional, Helix to Helix Collagen Cross-link: 
Stereospecific Collagen Molecular Structure In 
Type I Skin Fibrils" Biochemistry, Submitted for 
Publication (1986) 
Yamauchi, M., London, R.E., Guenat, C., Hashimoto, 
F., and Mechanic, G.L. "Structure and Formation of 
a Stable Histidine-based Trifunctional Cross-link 
in Skin Collagen J. Biol. Chem. In Press (1986) 
8 
. .  
G.L. Mechanic NAG 2-181 
Work to be Periormed 
January 1, lY87-becember 31, 19&7 
- -  
This year we shall be iinishing 02% a bone collagen 
turnover study perzormed on 55 r a t s  put on an experimental 
protocol that lasted iOr 6 months. The rats were treated as 
propoeed in our grant to model the weightless condition and 
had miniosmotic pumps implanted subcutaneously containing 
radioactive proline. In most case the miniosmotic pumps 
were of the 2 week variety, however, in the short time 
experimental periods there were pumps implanted that had a 
duration of 1 week. 
L 
We shall also be analyzing monkey bones rrom an 
immobilization study periormed by Dr. ljonald Young from 
which data was obtained for the nonmineralized and 
mineralized collagen (see publication above). 
Phosphoprotein in each OX the collagen compartments will be 
assessed. Pyridinoline cross-link will also be measured. 
Cross-links between phosphoprotein and collagen will be 
quantitatively determined in order Tor it to be fit into a 
model to be proposed zor 108s oi bone mass due to 
weightlessness. In addition bones from vitamin D deficient 
monkeys will be put through the same analytical procedures. 
The latter is an excelLent model f o r  osteopenia and 
osteomalacia. Using these models we shall be able to 
evaluate bone collagen structures in specific states ox 
disorder on a molecular and biochemical level. 
, .  - -  
DETAILED BUDGET NAG 2-181 
Budget Period: JANUARY 1, 1987 - 
F-E_R S 0 N N EL x 
G. L. Mechanic Prin. Invest. 20 
M. Yamauchi Co-Prin. Invest. 40 
Gloria Chandler Res. l’ech. I1 100 
Medical Insurance 5766IFTE 
Name Position Title Effort 
Totals 
DECEMBER 31, 1987 
- -  
Fringe 
SALARY Benefits TOTALS 
16,380 3,112 $19,492 
16,800 3,192 19,992 
20,496 3,894 24,390 
553,676 10,198 63,874 
1,226- 
Total 565,100 
EQUIPMENT. 
Shimazu HPLC Fluoresence Monitor plus Accessories 7,315 
SUPPLIES 
Radioactive NaBH. -1,600, L J  HILysine-l,500, CJH3Proline-l, 500 
Scintillation chemicals-2,000, Special chemicals(u1tra pure) 
2,200, Scintillation vials-1, 000, General Chemicals(buffers etc. ) 
1,200, Glassware-850, Chromatographic columns & Resins-3,000, 
Supplies for HPLC analyzers and sequencing peptides-2,100, 
Four cases of 0. 2p f ilterseS300ea. -1,200 18,150 
TRAVEL 
Two meetings per year per Investigator 2,400 
GTHER EXPENSES 
Cost of Publications- 900, Art work & Photography-800 
Photocopying costs-600, Communications-400, Books-250, 
Instrument Contract costs-1, 500 4,450 
TOTAL DIRECT COSTS $97,415 
Indirect Costs@25.0% Modified Total Direct Costs as per 
letter dated February 3, 1986 from Wayne R. Jones, Associate 
Vice Chancellor for Finance $22,525 
(Modified total direct costs=total direct costs less 
equipment) 
Gerald L. Mechanic, Ph.D. 
Contract Specialist Principal Investigator 


Biochemical changes in bone in a model of weightlessness

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