The Interaction(s) between Calf-Skin Hyaluronic Acid (Hyaluronan) and Dermal Type I Calf-Skin Collagen under 254 nm UV Radiation: Ability of Hyaluronan to Alter Qualitative and Quantitative Dimerization of Collagen Tyrosine Residues

The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs, providing not only essential physical scaffolding for the cellular constituents and initiating crucial biochemical and biomechanical cues, required for tissue morphogenesis, differentiation and homeostasis. Roughly divided into two groups, these are 1) the main fibrous ECM proteins: collagens, elastins, fibronectins and laminins. 2) Classification of proteoglycans (PGs) is based on their location and binding. Although many different molecular interactions are possible, they depend on the cells’ condition (i.e. “Normal”, Aged, Wounded/Fibrotic, and cancerous). There is little or no data that addresses the influence of the surrounding ECM on dityrosine formation. As a simpler model, we have replaced total PG with hyaluronan (HA) and have used purified calf-skin collagen tyrosine, which forms dityrosine (A2) under 254 nm UV in buffered solution and (near) physiological temperatures. Our results reveal a complicated temperature dependence involving factors relating to collagen HA structure, and collagen’s photochemical activation parameters.


Introduction
The extracellular matrix in mammalian dermis (ECM) is the non-cellular component, providing not only essential physical scaffolding for the cellular constituents and initiating crucial biochemical and biomechanical cues. In mammalian dermis, the predominant molecules are 1) Type I and Type III (85:15) collagens and 2) modular proteoglycans (PGs) (for review, see [1] [2]). Although many different types of molecular interactions are possible, they depend on the cells condition (i.e. "Normal", Aged, Wounded/Fibrotic, and Cancerous). Furthermore, PGs themselves appear to bind to many cell-surface receptors with high specificity, thereby activating signaling pathways that control cell proliferation, differentiation, adhesion, and migration.
Previous studies document that collagen and HA interact with each other in the ground state under physiologically relevant conditions in rabbit synovium, [3] [4] [5] [6], in vitro [5] and in aqueous solution [7]. Results of rotary shadowing electron microscopy and computer simulation indicate that HA self-aggregates into highly-branched networks that can form two-fold aggregation structures at the ends of the helix. HA mixed with collagen in situ causes a shift in distribution of fibrils to smaller diameters [3]. Hyaluronic acid (hyaluronan, HA) does not bind covalently to collagen, but collagen and HA do interact by mutual steric exclusion. This noncovalent interaction enables HA to form non-ionic complexes [7].
As the presence of dityrosine is diagnostic for protein damage, its presence in proteins has been proposed as a molecular probe of UV-induced photodimerization (reviewed in [8] [9]). There is little or no data that addresses the effect of the surrounding ECM on the photochemical production of dityrosine via excited state tyrosine (A*).
We have used a model in vitro buffered collagen/HA system to study the influence of HA molecules on UV-induced photodimerization. Shimazu [10] found that the rate of photo-dimerization of tyrosine to dityrosine in aqueous solution at small irradiation times is quasi-linear and proportional to the initial tyrosine concentration [A o ]. Our preliminary results with collagen and HA reveal a complicated temperature dependence involving several factors relating to structure and conformation of the collagen, the HA, as well as photochemical activation parameters.

Sample Characterization
Fluorescence Spectroscopy: Emission spectra of collagen samples were rec- R < 1 signifies that the control sample degrades faster than the test sample, which denotes relative stabilization of collagen by HA; R > 1 indicates destabilization. Rates of A 2 formation were calculated for given temperatures between T = 8˚C and T = 62.9˚C for collagen Coll(o) and Coll(HA) samples.

Results
Photolysis of Collagen and Collagen + HA residues at 254 nm produces A 2 dimers in qualitatively similar manner to Shimazu's results at room temperature [10]. Figure 1 shows that at short irradiation times, the initial formation of dityrosine is also linear with irradiation time, but the relative effect of HA depends on the temperature. At 8˚C, HA stabilizes the collagen polymer (R < 1; Figure 1(a)), whereas at 51˚C, HA destabilizes it (R > 1, Figure 1 [12]. At T < 10˚C, the collagen triple helix exists almost exclusively [12]. At 35˚C < T 50˚C stabilization by reversible predenaturational micro-unfolding of collagen "cooperative blocks" is significant and may result in the shape of the curve for collagen alone in Figure 2.

2)
Collagen + HA (White Dots): The addition of HA decreases the rate of dityrosine formation below Tm (36˚C); above Tm, HA increases the rate of dityrosine formation. At T < 50˚C the collagen configuration in the Collagen + HA system may be restricted by the interacting HA molecules. Therefore, at T > 35˚C, the overall temperature dependence of dityrosine formation may depend primarily on photochemical activation parameters.
With Collagen-HA, the overall rise in formation rate with at T > 35˚C may be attributed to a higher degree of micro-unfolding between 20˚C and 60˚C in Collagen-HA [12] [14], which favors photodimerization [12] [13] [14]. Open Journal of Physical Chemistry 50˚C, but increase below 10˚C. On the other hand, in Collagen + HA, the rate of dityrosine formation is a minimum from 8˚C -10˚C, and increases monotonically between 30˚C ≤ T ≤ 50˚C. At temperatures above 50˚C, both collagen samples start to denature and dissociate. The data for T < 20˚C for the collagen sample in the absence of HA is much less precise than at higher temperatures, resulting in a poor correlation coefficient (r 2 = 0.40) compared with r 2 = 0.77 for the companion collagen + HA sample. The resulting curve is more "well behaved" than those in Figure 2 and can be reasonably be described by a 2nd order linear regression curve (r 2 = 0.69). At T < 15˚C, R decreases markedly with rising temperature, having a value of 2.4 at 8˚C and decreasing to 1.4 at T = 20˚C. R ~ 1.2 between 25˚C and 40˚C. At temperatures between 40˚C ≤ T ≤ 60 ˚C, R < 1. Even here, however, the data tend to be scattered at T < 20˚C, although much less than in Figure 2).

Discussion
Previous literature has indicated that the collagen environment in the ground state is radically changed in the presence of HA [3] [4] [5] [6] [7]. Öbrink concluded from light scattering and turbidimetric studies under physiologic conditions that HA and collagen affect each other by a mutual steric exclusion [5]. In addition, there are several salient publications showing collagen-HA interactions in gels [4] [6]. In aqueous solution [7], there is visible evidence from electron microscopy that collagen increases the spacing between collagen in collagen HA co gels. Our results indicate a priori molecular interactions of collagen tyrosyl radicals in the excited state with ground state HA. Such a situation is, R < 1.00 indicates that HA retards dityrosine formation (i.e. increases stability); R > 1.00 indicates that HA increases the rate of dityrosine formation (i.e. decreases stability).
indeed, compatible with an intimate relationship between collagen and HA.
Our work demonstrates that collagen-bound tyrosine qualitatively behaves similarly to Shimazu's system in unbound tyrosine [10] [11] at physiological pH. Interactions between collagen and surrounding ECM could either facilitate or retard dimerization which results in the temperature-dependent shifts in R ratios, seen in Figure 2 and Figure 3. In the present case, the net result of collagen-HA interactions stabilizes tyrosyl residues by increasing its stability at temperature below ~36˚C. Our previous work [13] shows that Skh-1 hairless mouse skin has different fading properties in solution than the present calf skin sample. This suggests that these two collagen samples may have a different overall geometry and/or chemical state and that the tyrosyl residues may therefore interact differently with their surrounding environment. It should be pointed out that effects of surrounding environment on polymer stability and activity need not be the same [15].
Type I collagen and its fluorophore tyrosine are inherently unstable. At physiological temperatures collagen in solution can form aggregates, gels or slowly autoxidize. At higher temperature, it can also irreversibly decompose to gelatin, dissociate, and/or change its conformation from helix to random coil. Because of this instability, it is necessary to prepare matched Coll and Coll-HA solutions within one day of an experiment using the same collagen stock solution for both control and experimental samples to minimize possible artifacts. Figure 2 indicates that in the collagen-HA system, particularly at T < 50˚C the collagen configuration may be restricted by the interacting HA molecules. Therefore, the overall temperature dependence of dityrosine formation in the collagen alone system may depend primarily on photochemical activation parameters at T ≥ 35˚C. On the other hand, at T ≤ 10˚C, the triple helix exists almost exclusively, and is less stable than the (micro)unfolded state [12] [13] [14]. This loss of stability could increase the rate of dityrosine formation, and it may also contribute to the relative lack of precision in dimerization rate at temperatures below 20˚C (see Figure 2 and Figure 3). With Collagen-HA, the monotonic rise in formation rate with temperature at T ≥ 35˚C may be attributed to a higher population of excited state molecules, which favors photodimerization. At T > 60˚C, complete denaturation and dissociation to single coils ensue in both samples.
Collectively, the evidence seems to indicate that although there is no electrostatic interaction between collagen and HA, the two polymers are intimately involved with each other. This has the effect of stabilizing the collagen at a relatively wide range of temperatures. At higher temperatures, activation parameters play a more prominent role. At T > 60˚C, there is complete conversion of collagen to a random coil and subsequent degradation, and this markedly increases the rate of photodimerization for both samples. Open Journal of Physical Chemistry DOD Grant # 911 NF-10-1 0448. LaToya Freeman, and Ortega Edukye were first year medical students at MSM, and have since graduated. They received salary from MBRS #GM08248 at the Morehouse School of Medicine.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.