Transcript Julian M Menter

Histology of Normal Human Skin
DERMAL COLLAGENS
(1) “Structural” component of skin
(2) Comprises ~ 70 – 80 % of dermis
(3) Mostly Type I and Type III in humans
Some properties of skin collagens
• Skin collagens belong to a genetically distinct
group of structural proteins.
• Type I and type III collagens are the
predominant collagens in the dermis, in the
ratio ~ 85:15.
• In this work, we focus on Type I collagen
Structural Aspects of Type I Collagen (1)
Tropocollagen is the basic structural unit of skin
collagen; it consists of 2 α1(I) chains and
1 α2(I) chain. These chains have a left –
handed helical structure, and they are wound
in a right – handed superhelix.
It has the general formula X – Y – Gly, and
contains ~10 – 15% each of Proline and
Hydroxyproline
Structural Aspects of Type I Collagen (2)
Tropocollagen forms fibrils by cross – linkage with
other tropocollagen molecules.
These “normal” linkages can involve one of several
molecules, but the “fundamental” linkage involves
the formation of Schiff base between free amino
groups of (hydroxy)lysine and nearby aldehydes
formed by lysyl oxidase on some of the
(hydroxy)lysine residues
See JP Bentley J Invest. Dermatol. 73: 80 – 83, 1979
Schematic representation of the formation of intermolecular cross-links of collagen.
Telopeptides are Non – Helical Portions and the N- and
C- Terminal Ends of the Collagen Molecule
• (a) Telopeptides have an antiparallel β – pleated sheet*
• (b) Telopeptides are high in tyrosine and phenylalanine
residues, low in arginine and (hydroxy)lysine residues**
• (c) Tyrosine residues in favorable position to form dimers,
“excimers” and higher oxidation products*
• (d) Telopeptides (Mainly N- telopeptides) are necessary for
fibrillogenesis
•
•
* D. Helseth et al, Biopolymers 18 3005 – 3014 (1979)
* *A.L. Rubin et al, Science 139 37 – 38 (1963)
Proposed Structure of Collagen N-Telopeptide
Note that the hydrophobic AA face in towards the center of the molecule and the
hydrophilic AA face out towards the surface
Schematic representation of proposed secondary structure of N - telopeptide
Tyrosine Can be Degraded by
Oxidation or UV radiation. Degraded
Collagen Has Different Fluorescence
Properties than Normal Collagen
Spectral Differences in Calf Skin
Collagen as Function of Age
Lot# 159 “New” (obtained Feb
2012 and used July 2012)
80
Wavelength (nm) vs Icorr#121
Wavelength (nm) vs Icorr#159
60
40
20
0
300
320
340
360
380
Wavelength (nm)
400
420
440
Corrected Fluorescence Intensity (a.u.)
Corrected Fluorescence Intensity (a.u.)
Lot# 121 (5 years old; kept in
refrigerator in dark at 4o C)
80
60
Wavelength (nm) vs Icorr#159
Wavelength (nm) vs Icorr#121
40
20
0
300
320
340
360
380
Wavelength (nm)
400
420
440
Fluorophores Likely to Exist in “Aged”
Collagen
•
•
•
•
•
•
Putative Compound
Exc (nm) Fluor (nm)
(1) Tyrosine
275
305
(2) DOPA
285
330
(3) Dityrosine
325
400
(4) DOPA oxidation prod. a * ~ 350 ~ 450
b ** ~285 360
• (5)***Advanced Glycation Endproducts ~330-360, ~ 440-450
•
•
*Unstable to O2
• ** Photolabile to 254 nm UV
*** Schiff Base between glucose and free -NH2 of lysine
Tyrosine Dimerization to Dityrosine
Decreased in Autoxidized Collagen
• Fresh Collagen was
irradiated with UVC (254
nm) as a function of time.
• Black Dots - “aged”
collagen sample
• White Dots - fresh collagen
sample
•
See O Shimizu Photochem Photobiol 18(3)
123 – 133, 1973
Kinetics of dityrosine formation
• Reaction
• (1) A + hγ ----> A*
(2) A* ----> A + Δ
• (3) A* + A ----> A – A
•
(a) Rx (3) is slow step in UV
photodimerization.
•
(b) System (3) must “build up” to a
steady – state that requires t > 0
•
(c) As t ----> 0 , rate of A-A formation
is maximum; dA*/dt ~ k[A] and is
quasi - linear (A* is << A)
•
•
•
•
•
Rate
Iabs Φrx = k*[A]
- kT [A*]
- kAA [A][A*]
Photodestruction of DOPA Oxidation Product is
Increased in Autoxidized Collagen
• Oxidized Sample “aged”
in dark at 4 C for ~ 5
years was irradiated with
UVC (254 nm) as a
function of time
• Black Dots – “aged”
collagen sample
• White Dots - new
collagen sample
Kinetics of “DOPA – Oxidation Product” Photo Destruction
• Rate of “product”
disappearance is
• 1/F(t) = (slope)t + 1/F(0)
proportional to the
square of the
• “Plot 1/F, proportional
“product”concentration
to 1/[AO], vs. time;
.
should get a straight
line whose slope is
• This suggests a “double
proportional to rate
molecule” which
constant”
disappears on UV
irradiation
Does the Surrounding Extracellular
Matrix Have an Effect on Tyrosine
Spectroscopy and/or Photochemistry?
Extracellular Matrix Proteins
•
Supplies the support system for dermal cells
• Consist largely of
• (1) Hyaluronic Acid (HA; Hyaluronan) – Major component.
• (2) Sulfated Glycosaminoglycans (GAGS).
• (3) Proteoglycans
• (4) Elastin
• (5) “Others”
DISTRIBUTION OF GAGS IN SKIN
(Uronic Acid equivalents/mg)
• [DERMIS]
•
•
•
•
•
•
(pmole UA/mg
HA
HepSO4
DS
C4/6S
Heparin
• [EPIDERMIS]
total wet skin)
243.28
22.25
170.30
71.95
5.18
•
•
•
•
•
•
(pmole UA/mg total wet skin)
HA
0.48
HepSO4
0.44
DS
0.56
C4/6S
0.50
Heparin
0.22
DERMAL PROTEOGLYCAN AGGREGATE
DERMAL PROTEOGLYCAN AGGREGATE
“First Approximation” to In Vivo Milieu
(1) Collagen – Hyaluronic acid
mixtures represent a first
approximation to the in vivo
dermal environment
(2) Effect of other molecules in the
proteoglycan not taken into
account
Interactions among HA, Collagen , and
HA – Collagen Mixtures
• Nagorski et al studied HA-Collagen interactions via measurements of the
polymers in polyacryamide gels. They concluded that:
•
(1) The interactions among collagen monomers are largely hydrophobic.
•
(2) The interactions among HA molecules involve H – bonding and repulsive ion
forces
•
(3) The interactions among molecules in the collagen – HA mixture indicate “either
a hydrophobic or an attractive interaction between segments of the two
biopolymers”.
•
(4) These workers favor the latter theory.
•
C. Nagorski et al Res. Commun Mol Pathol Pharmacol (1995) 89(2): 179 - 188
So….Does the Surrounding
Extracellular Matrix Have an Effect on
Tyrosine Spectroscopy and/or
Photochemistry?
Hyaluronate Has Only Marginal Effect on Fluorescence
Fading of DOPA – Oxidation Product
(1) Curved regression line in
Arrhenius plot reflects collagen
change of phase (melting) at ~ 36 C
(2) There is no statistical difference
between Collagen alone and
Collagen + Added HA at the 95%
confidence level
Hyaluronate Has a Variable Effect on Photooxidized
DOPA Derivative on Collagen I
Fading of Calf Skin Collagen Fluorescence •
under 254 nm UV in 0.1 M PO4 Buffer,
pH = 7.4
• (2) In this experiment, HA seems
to facilitate fluorescence fading
after extensive photolysis
o
T = 16.0 C ex = 270 nm
em = 360 nm
Black Dots = Collagen Alone
White Dots = Collagen + HA (1:2)
Reciprocal Normalized Fluorescence
1 / In(270)360)
(1) Effect is small; inconsistent
2.5
• (3) But effect is probably not
significant
2.0
1.5
• (4) Apparent “stability” of
collagen could reflect “back”
reactions.
TIME (min) vs 1/In(270)360
TIME (min) vs In(270)360collHA
Plot 1 Regr
1.0
0.5
0
50
100
150
Time (min)
200
250
•
Hyaluronate Has a Variable Effect on
Photooxidized DOPA Derivative on Collagen II
• (1) Effect is Small
Fading of 270/360 nm Collagen
Fluorescence under 254 nm UV
o
T = 25 C
ex = 270 nm
em = 360 nm
Black - Collagen Alone
White - Collagen + HA (1:2)
• (2) Effect is inconsistent
Data of 24 - 25 October, 2010
3.5
• (3) In this experiment, HA
seems to retard
fluorescence fading
3.0
2.5
2.0
1.5
Time (min) vs 1/In(270)360 coll
Time (min) vs 1/In(270)360 collHA
Plot 1 Regr
1.0
• (4) But effect is probably
not significant
0.5
0
50
100
150
200
Time (min)
250
300
350
Hyaluronate May Have a Small Effect
on Dityrosine Formation I
Build-up of Dityrosine Fluorescence
o
T = 20 C
ex = 325 nm
em = 400 nm
Black = Collagen Alone
White = Collagen + HA 1:2
• (1) Build-up of
dityrosine is slightly
retarded by HA
Normalized Fluorescence at (325)400 nm
Data of 23 August, 2010
2.5
• (2) May be significant
2.0
1.5
TIME (min) vs In(325)400
TIME (min) vs In(325)400 HA
Plot 1 Regr
1.0
0.5
0
50
100
150
200
Time (min)
250
300
350
Hyaluronate May Have a Small Effect
on Tyrosine Photodimerization II
Build-Up of 325/400 nm Fluorescence in
Collagen + HA under 254 nm UV
o
T = 20 C
Black - Collagen Alone
White - Collagen + HA (1:2)
Normalized Fluorescence [In(325)400]
Data of 24 October, 2010
• (1) Build-up of
dityrosine is slightly
retarded by HA
2.5
• (2) May be significant
2.0
1.5
TIME (min) vs In(325)400
TIME (min) vs In(325)400HA sh
Plot 1 Regr
1.0
0.5
0
50
100
150
Time (min)
200
250
300
WHY?
• (1) HA solution is more viscous than H2O (Could lower rate of
tyrosine dimerization, since two tyrosine molecules must diffuse
towards each other
• (2) Based on previous studies, one might expect interactions
between Collagen and HA. The interactions among molecules in the
collagen – HA mixture indicate “either a hydrophobic or an
attractive interaction between (ionic) segments of the two
biopolymers”
•
C Nagorski et al, Research Communications in Mol Pathology and Pharmacology
89(2) 179 – 188 (1995)
POSSIBLE REASONS (HYPOTHESIS)
• (1) Photochemical intermediate, tyrosyl
radical, may not be sensitive to ionic
processes. This favors hydrophobic processes
• (2) Tyrosyl residues in telopeptide are in
hydrophobic region of collagen molecule and
not readily accessible to HA
CONCLUSIONS
• (1) Hyaluronate has a small, if any, effect on
the spectroscopy and photochemistry of
collagen fluorophores
• (2) Reasons not known, but we hypothesize
that hydrophobic tyrosine residues in the
telopeptide may be shielded from the
extracellular matrix.
Acknowledgements
• This work was supported in part by DOD Grant
#911 NF-10-1, MBRS Grant #GM08248, and
RCMI Grant # RR 3034. There are no conflicts
of interest.
ACKNOWLEDGEMENTS
•
•
•
•
Abrienne M Patta
Latoya Freeman
Otega Edukuye
Imani Herbert