Pep-1 as a Novel Probe for the In Situ Detection of Hyaluronan
Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas
Correspondence to: Dr. Mark E. Mummert, Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9069. E-mail: mark.mummert{at}utsouthwestern.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: hyaluronan hyaluronan-binding protein Pep-1 extracellular matrix
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cutaneous HA metabolism is normally a tightly regulated process. However, abnormally high concentrations of HA in the skin have been reported for a number of diseases including psoriasis (Wells et al. 1991; Tammi et al. 1994
), scleroderma (Sondergaard et al. 1997
; Passos et al. 2003
), amyotrophic lateral sclerosis (Ono et al. 1996
), lichen sclerosus et atrophicus (Kaya et al. 2000
), and acute cutaneous graft vs host disease (GVHD) (Milinkovic et al. 2004
). Similarly, pharmacologic agents such as glycolic acid (Bernstein et al. 2001
), substance K (Kim et al. 2004
), and estrogens (Gendimenico et al. 2002
) have been shown to markedly increase HA expression in the skin. In contrast, diabetic patients with severely restricted joint mobility of the hands showed a significant loss of HA in the basement membrane zone of the skin (Bertheim et al. 2002
), whereas topical application of steroids also reduced HA expression (Zhang et al. 2000
). Thus, enhanced or reduced concentrations of HA may contribute to the dermatopathologies of various diseases as well as treatments. Lastly, HA may provide a useful marker to monitor disease progression.
Analyses of the concentration and polymer size of HA are typically determined by biochemical methods such as chromatography (Okamoto et al. 1999; Chaidedgumjorn et al. 2002
; Karlsson and Bergman 2003
), gel electrophoresis (Ikegami-Kawai and Takahashi 2002
), and ELISA-like methods (Stern and Stern 1992
). On the other hand, HA localization studies are based primarily on histology. Classic histochemical studies of HA are based on nonspecific cationic dyes such as Alcian blue. Ripellino et al. (1985)
developed a more specific technique for staining HA in brain sections using the HA-specific probe, HA-binding protein (HABP). HABP is a complex of aggrecan and link protein and is derived from bovine cartilage (Tengblad 1979
). Tammi and colleagues (1988)
evaluated the distribution of HA in human skin using HABP as their probe. They found weak dermal staining but intense staining in the basal and spinous layers of the epidermis. Others have reported that HABP more strongly stained the dermis compared with the epidermis (Wells et al. 1991
; Lin et al. 1997
). Differences in the epidermal staining pattern have also been reported, with weak reactivity in the basal layer and more intense staining in the spinous and granular layers (Lin et al. 1997
). Discrepancies in HABP staining profiles make comparisons among studies difficult to interpret and suggest the need to standardize current techniques and/or develop alternative probes for HA.
Recently, we have developed a HA-binding peptide (termed Pep-1) using the phage display technique (Mummert et al. 2000). Analyses of the Pep-1 amino acid sequence showed that it did not resemble known HA-binding domain sequences of CD44 or the B(X)7B motif, where B represents a basic amino acid residue and X any non-acidic residue (Entwistle et al. 1996
; Mummert et al. 2000
). Alanine scanning studies showed that the critical amino acids for HA binding were contiguous residues at positions 4, 5, and 6 (W, Q, F) and positions 9, 10, and 11 (L, T, V) (Mummert et al. 2000
). Finally, in vitro studies showed that Pep-1 binds HA in immobilized, cell-associated, and soluble forms. Thus, we decided to investigate the potential utility of Pep-1 to serve as a probe for the qualitative and quantitative assessment of HA in the skin. Herein, we report the HA staining profiles of Pep-1 and HABP in human foreskin and mouse ear skin.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peptides and HABP
Pep-1 (GAHWQFNALTVR) and a scrambled peptide control (WRHGFALTAVNQ) were synthesized by Invitrogen (Carlsbad, CA) using standard fMOC chemistry as previously described (Mummert et al. 2000). Briefly, an amidated and biotinylated lysine residue was included at the C terminus of the linker sequence (GGGS) to facilitate detection of peptides. Stock solutions were prepared by dissolving peptides to 1 mg/ml in dH2O and were stored at 20C. HABP was purchased from Seikagaku Corporation (Tokyo, Japan) and biotin labeled with N-hydroxysuccinimido-biotin (Pierce Chemical Co; Rockford, IL) as described by the manufacturer.
Sample Preparation and Staining Procedure
Mouse ear skin and human foreskin were embedded in Optimal Cutting Temperature compound (OCT; Sakura Finetek, Torrance, CA), snap frozen in liquid N2, and sectioned at 8-µm thickness. Skin samples were fixed for 10 min in acetone and blocked for 10 min in 100 mM glycine followed by a 30-min incubation with PBS containing 1% BSA. Slides were washed with PBS and treated for 16 hr at 4C with Streptomyces hyaluronidase at 100 U/ml (Sigma-Aldrich; St Louis, MO) or mock treated with buffer alone (100 mM sodium acetate, pH 5.0). Biotinylated Pep-1 (5 µg/ml), scrambled peptide control (5 µg/ml), or HABP (1 µg/ml) in PBS containing 1% BSA were incubated on slides for 5 hr at 37C. In some experiments, biotin-conjugated peptides were used at 50 µg/ml to ascertain the impact of peptide concentration on the HA staining patterns. After washing in PBS containing 0.05% Tween 20, slides were developed by a 30-min incubation with fluorescein 5 isothiocyanate (FITC)-conjugated streptavidin (Jackson ImmunoResearch Laboratories; West Grove PA) diluted 1:200 in PBS containing 1% BSA. Skin sections were evaluated under an Olympus BX60 fluorescence microscope (Olympus; Melville, NY) equipped with a Sensys digital camera system (Photometrics; Tucson, AZ) and Metaview software (Universal Imaging Corp.; Downington, PA).
Glycosaminoglycan Inhibition Assay
To determine the specificity of Pep-1 for HA moieties in skin, we tested the inhibitory potential of HA oligomers and polymers as well as two HA-related glycosaminoglycans (chondroitin sulfate A and dermatan sulfate). Briefly, biotinylated Pep-1 (5 µg/ml) was incubated overnight in the presence of HA polymers, HA oligomers, chondroitin sulfate A, or dermatan sulfate in PBS containing 1% BSA at 37C. All of the inhibitors were used at a concentration of 1.5 mg/ml. Peptide solutions containing the various glycosaminoglycan inhibitors were then added to skin sections and developed as above.
Other Fixation Techniques
Mouse ear skin was embedded in OCT, snap frozen in liquid N2, and sectioned at 8-µm thickness. Skin samples were fixed for 10 min in PBS containing 3% paraformaldehyde and 0.5% cetylpyridinium chloride (CPC). After washing in PBS, sections were blocked for 10 min in 100 mM glycine followed by a 30-min incubation with PBS containing 1% BSA. Staining of paraformaldehyde/CPC-fixed samples was performed exactly as described for acetone fixation.
Permanent sections were prepared by embedding acetone-fixed ear skin in paraffin. Sections were cut to 8-µm thickness, deparaffinized, blocked with glycine and BSA as above, and stained with Pep-1 or the scrambled peptide after pretreatment with Streptomyces hyaluronidase or mock treated with buffer alone. Skin sections were stained with Pep-1 and the scrambled peptide control as above.
Quantitative Analysis
Integrated fluorescence intensities in 2030 fields per skin section (n=3) were obtained with Metaview software (Universal Imaging Corp.) from images captured at x200 magnification. Mean values were compared with a two-tailed Student's t-test and differences considered significant for p<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that HABP stained the dermis of human foreskin uniformly and with high intensity (Figure 1). In contrast, HABP predominantly stained HA in the spinous and granular layers of the epidermis, whereas staining in the basal layer was relatively weak or absent. No staining was detected in the stratum corneum. Quantitative analyses showed insignificant differences (p>0.05) between the dermal and epidermal (spinous and granular layers) integrated fluorescence intensities (Figure 2). Pretreatment of cryostat sections with Streptomyces hyaluronidase, an enzyme that specifically degrades HA, significantly (p<0.01) reduced HABP staining in both the epidermis and dermis, showing HA specificity (Figure 1 and Figure 2). Unlike HABP, Pep-1 showed diffuse and relatively weak staining of HA in the dermal compartment (Figure 1). With the exception of the stratum corneum, Pep-1 stained all of the epidermal layers, although the intensity of the staining appeared to be in the order basal layer > spinous layer > granular layer. However, integrated fluorescence intensities were not significantly different among the various epidermal layers as assessed by ANOVA (p>0.05) (data not shown). On the other hand, comparisons of integrated fluorescence intensities between the epidermis and dermis showed that Pep-1 stained the epidermis significantly better than the dermis (Figure 2; p<0.01). We next evaluated the specificity of Pep-1 staining by pretreatment of slides with Streptomyces hyaluronidase. Qualitative (Figure 1) and quantitative (Figure 2) analyses showed that digestion of HA from skin sections nearly completely abrogated Pep-1 staining, indicating HA specificity (Figure 1 and Figure 2).
|
|
HABP stained the dermis of mouse ear skin uniformly and with high intensity (Figure 3 and Figure 4). On the other hand, HABP stained the epidermis mainly in the stratum corneum and basal lamina with relatively weak or no binding in the vital epidermis. Pretreatment of slides with Streptomyces hyaluronidase significantly reduced the intensity of staining in the dermis, whereas the stratum corneum and basal lamina was partially resistant to enzymatic digestion (Figure 3 and Figure 4). Finally, preincubation of HABP with HA polymers (1.5 mg/ml) significantly reduced HA staining, further showing the specificity of HABP for HA (data not shown).
|
|
Effect of Pep-1 Concentration on HA Staining Patterns
Because the observed staining pattern of Pep-1 could have been the result of the peptide concentration, we assessed the impact of higher Pep-1 concentrations on the staining profiles in human and mouse skin. As shown in Figure 5, a 10-fold higher concentration (50 µg/ml) of Pep-1, but not the scrambled peptide control, markedly enhanced the fluorescence intensities in both human and mouse skin. However, higher concentrations of Pep-1 did not result in a HABP-like staining pattern. These results show that Pep-1 staining profiles are not concentration dependent.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In an effort to develop alternative HA probes for histology, we have compared the staining profiles of HABP and Pep-1 in human and mouse skin. Perhaps the most striking finding was that HABP uniformly stained the dermis with high intensity, whereas Pep-1 showed diffuse dermal staining with relatively weak intensity after acetone fixation. In contrast, fixation of tissues with paraformaldehyde/CPC resulted in intense dermal staining for both HABP and Pep-1.
How can we explain the discordant staining profiles observed in these studies? One possible reason for differences in dermal staining between HABP and Pep-1 after acetone fixation is that Pep-1 may have a markedly slower diffusion rate in the dermal compartment compared with HABP. However, given the relatively small size of Pep-1 compared with HABP, one would expect Pep-1 to more easily diffuse into the tissue for staining, not vice versa.
Recent biophysical studies have shown that HA adopts distinct conformations based on polymer length (Scott and Heatley 2002), degree of hydration (Cowman et al. 2005
), and its association with membrane surfaces (Ionov et al. 2004
). Although biophysical analyses have been based exclusively on in vitro systems, Milinkovic et al. (2004)
have evaluated the adhesion of non-stimulated lymphocytes to skin biopsies from patients with acute GVHD. Although HA was expressed throughout GVHD skin (i.e., epidermis and dermis), the lymphocytes bound almost exclusively to HA expressed on endothelial cells of dermal vessels. These authors concluded that HA expressed by dermal endothelium was specialized to support lymphocyte adherence under sheer stress (Milinkovic et al. 2004
). We suggest that differences between HABP and Pep-1 staining profiles may reflect differences in HA tertiary structures and/or HA lattice meshwork in different layers of the skin.
Can differences in HA conformations explain why Pep-1 stains the dermis after paraformaldehyde/CPC fixation? Recently, Cowman et al. (2005) evaluated the conformations of HA by tapping mode atomic force microscopy. When HA was deposited on the structured water layer of prehydrated mica, an extended conformation was favored. In contrast, HA deposited on mica lacking structured water favored a weakly helical, coiled conformation. Thus, the degree of hydration significantly impacts the conformation of HA polymers. Because precipitation likely alters the hydration of HA polymers in situ, we suggest that precipitated dermal HA (paraformaldehyde/CPC fixed) adopts a different conformation than non-precipitated dermal HA (acetone fixed). Interestingly, Streptomyces hyaluronidase abrogated dermal staining by HABP after acetone fixation but only partially reduced dermal HABP staining after paraformaldehyde/CPC fixation. Failure of Streptomyces hyaluronidase to completely remove HA staining for both HABP and Pep-1 may suggest that hyaluronidase cannot digest all HA conformations and/or lattice meshwork assemblies.
In terms of utility, Pep-1 may allow expression of epidermal HA to be studied in a number of pathologic states in fine detail after acetone fixation. Pep-1 may be of special value for studying epidermal HA expression in experimental conditions in mice, where the thin epidermis can be especially difficult to distinguish from the underlying dermis. Lastly, comparisons of Pep-1 and HABP staining profiles in conjunction with biophysical and biochemical analyses may shed new light on the relationship between HA structure and function in situ. Based on our experimental observations, we suggest that Pep-1 may represent a unique reagent to probe HA conformation.
Finally, we should emphasize that the synthetic nature of Pep-1 may represent an advantage over HABP, which is derived from a complex biological material (i.e., cartilage) (Tengblad 1979). For example, Armstrong and Bell (2002)
showed that the fraction of link proteins in HABP preparations can significantly impact the size of the HA polymers detected in solution. As a synthetic peptide, it should be possible to synthesize Pep-1 in a more reproducible fashion, which may reduce experimental variation.
![]() |
Acknowledgments |
---|
We would like to thank Patricia Adcock for her secretarial assistance and Dr. Akira Takashima for his thoughtful comments and suggestions.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armstrong SE, Bell DR (2002) Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis. Anal Biochem 308:255264[CrossRef][Medline]
Bernstein EF, Lee J, Brown DB, Yu R, Van Scott E (2001) Glycolic acid treatment increases type I collagen mRNA and hyaluronic acid content of human skin. Dermatol Surg 27:429433[CrossRef][Medline]
Bertheim U, Engstrom-Laurent A, Hofer PA, Hallgren P, Asplund J, Hellstrom S (2002) Loss of hyaluronan in the basement membrane zone of the skin correlates to the degree of stiff hands in diabetic patients. Acta Derm Venereol 82:329334[CrossRef][Medline]
Chaidedgumjorn A, Suzuki A, Toyoda H, Toida T, Imanari T, Linhardt RJ (2002) Conductivity detection for molecular mass estimation of per-O-sulfonated glycosaminoglycans separated by high-performance size-exclusion chromatography. J Chromatogr A 959:95102[CrossRef][Medline]
Cowman MK, Spagnoli C, Kudasheva D, Li M, Dyal A, Kanai S, Balazs EA (2005) Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy. Biophys J 88:590602
Entwistle J, Hall CL, Turley EA (1996) HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem 61:569577[CrossRef][Medline]
Fraser JR, Laurent TC, Laurent UB (1997) Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 242:2733[CrossRef][Medline]
Gendimenico GJ, Mack VJ, Siock PA, Mezick JA (2002) Topical estrogens: their effects on connective tissue synthesis in hairless mouse skin. Arch Dermatol Res 294:231236[Medline]
Gerdin B, Hallgren R (1997) Dynamic role of hyaluronan (HYA) in connective tissue activation and inflammation. J Intern Med 242:4955[CrossRef][Medline]
Hayen W, Goebeler M, Kumar S, Riessen R, Nehls V (1999) Hyaluronan stimulates tumor cell migration by modulating the fibrin fiber architecture. J Cell Sci 112(Pt 13):22412251
Ikegami-Kawai M, Takahashi T (2002) Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity. Anal Biochem 311:157165[CrossRef][Medline]
Ionov R, El Abed A, Goldmann M, Peretti P (2004) Interactions of lipid monolayers with the natural biopolymer hyaluronic acid. Biochim Biophys Acta 1667:200207[Medline]
Karlsson G, Bergman R (2003) Determination of the distribution of molecular masses of sodium hyaluronate by high-performance anion-exchange chromatography. J Chromatogr A 986:6772[CrossRef][Medline]
Kaya G, Augsburger E, Stamenkovic I, Saurat JH (2000) Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus et atrophicus. J Invest Dermatol 115:10541058[CrossRef][Medline]
Kim S, Kang BY, Cho SY, Sung DS, Chang HK, Yeom MH, Kim DH, et al. (2004) Compound K induces expression of hyaluronan synthase 2 gene in transformed human keratinocytes and increases hyaluronan in hairless mouse skin. Biochem Biophys Res Commun 316:348355[CrossRef][Medline]
Lin W, Shuster S, Maibach HI, Stern R (1997) Patterns of hyaluronan staining are modified by fixation techniques. J Histochem Cytochem 45:11571163
Milinkovic M, Antin JH, Hergrueter CA, Underhill CB, Sackstein R (2004) CD44-hyaluronic acid interactions mediate shear-resistant binding of lymphocytes to dermal endothelium in acute cutaneous GVHD. Blood 103:740742
Mummert DI, Takashima A, Ellinger L, Mummert ME (2003) Involvement of hyaluronan in epidermal Langerhans cell maturation and migration in vivo. J Dermatol Sci 33:9197[CrossRef][Medline]
Mummert ME, Mohamadzadeh M, Mummert DI, Mizumoto N, Takashima A (2000) Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking. J Exp Med 192:769779
Okamoto I, Kawano Y, Matsumoto M, Suga M, Kaibuchi K, Ando M, Saya H (1999) Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the Rho family of small G proteins. J Biol Chem 274:2552525534
Ono S, Imai T, Yamauchi M, Nagao K (1996) Hyaluronic acid is increased in the skin and urine in patients with amyotrophic lateral sclerosis. J Neurol 243:693699[CrossRef][Medline]
Passos CO, Werneck CC, Onofre GR, Pagani EA, Filgueira AL, Silva LC (2003) Comparative biochemistry of human skin: glycosaminoglycans from different body sites in normal subjects and in patients with localized scleroderma. J Eur Acad Dermatol Venereol 17:1419[CrossRef][Medline]
Ripellino JA, Klinger MM, Margolis RU, Margolis RK (1985) The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain. J Histochem Cytochem 33:10601066[Abstract]
Sakai S, Yasuda R, Sayo T, Ishikawa O, Inoue S (2000) Hyaluronan exists in the normal stratum corneum. J Invest Dermatol 114:11841187[CrossRef][Medline]
Scott JE, Heatley F (2002) Biological properties of hyaluronan in aqueous solution are controlled and sequestered by reversible tertiary structures, defined by NMR spectroscopy. Biomacromolecules 3:547553[CrossRef][Medline]
Sondergaard K, Heickendorff L, Risteli L, Risteli J, Zachariae H, Stengaard-Pedersen K, Deleuran B (1997) Increased levels of type I and III collagen and hyaluronan in scleroderma skin. Br J Dermatol 136:4753[CrossRef][Medline]
Stern M, Stern R (1992) An ELISA-like assay for hyaluronidase and hyaluronidase inhibitors. Matrix 12:397403[Medline]
Tammi R, Paukkonen K, Wang C, Horsmanheimo M, Tammi M (1994) Hyaluronan and CD44 in psoriatic skin. Intense staining for hyaluronan on dermal capillary loops and reduced expression of CD44 and hyaluronan in keratinocyte-leukocyte interfaces. Arch Dermatol Res 286:2129[CrossRef][Medline]
Tammi R, Ripellino JA, Margolis RU, Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Dermatol 90:412414[CrossRef][Medline]
Tengblad A (1979) Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding properties from cartilage. Biochim Biophys Acta 578:281289[Medline]
Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, et al. (2002) Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 195:99111
Termeer CC, Hennies J, Voith U, Ahrens T, Weiss JM, Prehm P, Simon JC (2000) Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 165:18631870
Wells AF, Lundin A, Michaelsson G (1991) Histochemical localization of hyaluronan in psoriasis, allergic contact dermatitis and normal skin. Acta Derm Venereol 71:232238[Medline]
Yagi R, Nagai H, Iigo Y, Akimoto T, Arai T, Kubo M (2002) Development of atopic dermatitis-like skin lesions in STAT6-deficient NC/Nga mice. J Immunol 168:20202027
Zhang W, Watson CE, Liu C, Williams KJ, Werth VP (2000) Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy. Biochem J 349:9197[CrossRef][Medline]