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Biochemistry of hair protein
Biochemistry of Hair Protein
3 Howard P. Baden, MD
From the Department of Dermatology, Harvard Medical School, and Massachusetts General Hospital, Boston, Massachusetts
The hair shaft is surrounded by a layer of overlapping cuticle cells that are resistant to chemical and mechanical injury. These cells protect the cortex, which makes up the bulk of the hair shaft. The center of large hairs contains a medulla that is similar in composition to the inner root sheath but also has pigment granules. The fibrous character of hair results from the structural proteins in the cortical cell. Research on the chemistry of hair has been primarily directed toward studying these proteins. Various techniques have been used to isolate, purify, and characterize them.‘-3 The cortical proteins were found to be insoluble in neutral buffers and extractable with a minimum of degradation by using alkaline denaturing solvents containing a disulfide-reducing agent. Urea is a commonly used denaturingagent, and mercaptoethanol or dithiothreitol, or both, the reducing agents. In order to keep the proteins from aggregating, the cysteine residues were converted to S-carboxymethyl groups with iodoacetic acid. The protein solubilized from human hair is of two major classes: fibrous proteins that make up the filaments observed by electron microscopy and matrix proteins in which they are embedded.4-7 Electrophoretic and chromatographic studies show that both proteins contain a number of components that differ in size and chemical composition, although members of one class tend to be more similar to one another than to components from the other class. The major differences between the classes of proteins are their helical and cystine contents (Table 3-l). The fibrous protein had a helical content of 50% and contained 6 residues of )/zcystine per 100 amino acid residues, whereas the matrix proteins were nonhelical and had 25 to 30 residues of )/zcystine per 100 amino acid residues. An earlier technique used for separation was polyacrylamide gel electrophoresis in the presence of 6 M urea. This technique showed that the fibrous protein had four or more components, whereas the matrix proteins appeared as a single band (Fig. 3-l). When the electrophoresis was done at high gel densities, which is more effective in separating low-molecular weight proteins, a number of matrix proteins were visualized and the heterogeneity of the matrix proteins was better appreciated.8 The heterogeneity of the fibrous and matrix proteins was best demonstrated by two-dimensional electrophoresis using charge separation in the presence of urea in the first direction and size separation by SDS polyacrylamide gel electrophoresis in the second 22
October-December Volume 6 Number 4
1988 Biochemistrv
23
of Hair Protein
FIG. 3-1. Disc electrophoresis of the S-carboxymethyl hair proteins in 6 M urea. F is the fibrous polypeptides and M the matrix band. The arrow indicates the direction of electrophoresis.
direction.9 The matrix proteins proved to be even more complex when they were studied by chromatographic techniques. As a result of the work on the epidermal fibrous proteins, there was an interest in examining the fibrous proteins of hair in a more native state. The fibrous proteins exist in a reduced form in the lower hair shaft and become cross-linked by disulfide bonds when the cortical cells die and, of course, remain that way in the hair shaft. The major reason for studying the native proteins is that there are varying degrees of posttranslational modification of protein in cornified cells, and a clearer picture of the fibrous proteins could be obtained by studying them in their native reduced state. In order to isolate the native fibrous proteins of hair, the cortex of plucked hairs was dissected free of the inner and outer root sheaths. The tissue was homogenized, washed with water, and extracted either with a urea or SDS buffer. The protein extract was analyzed by two-dimensional electrophoresis, and basic and acid classes of fibrous polypeptides were found. There were four components in each, and they differed in mobility and size from the acidic and basic keratins of epidermis (Fig. 3-2).l’JBy isolating messenger RNA from the cortical cells, it was possible to show that they were all direct translational products and not derived one from the other by breakdown or posttranslational modification. Extraction of hair protein from the hair shafts without S-carboxymethylation followed by two-dimensional electrophoresis showed proteins similar to the native proteins described above,” but fewer individual components were identified and they differed somewhat in molecular weight and perhaps isoelectric points.
FIG. 3-2. Schematic diagram of two-dimensional electrophoresis of human epithelial (0) and hair (a) cytokeratins. A, P, and Bare standards. The numbers refer to the Moll17 catalog. (From Heid et al.,*0 with permission).
These proteins very likely correspond to the eight S-carboxymethyl polypeptides identified in wool, but direct comparison is not possible because of the modification induced by the Scarboxymethylation.12 Nevertheless, these reTABLE 3-1. Amino Acid Analyrls of the Alpha Fibrous (Alpha) and Matrix S-carboxymethyl Protolns of Halr. Data are Expressed as Residues per 100 ResiduesAmlno Adds
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine S-carboxymethyl cysteine
Alpha
Matrix
2.9 0.6 7.0 8.4 5.9 10.5 17.1 5.6 5.3 6.0 5.3 0.6 3.0 9.8 2.8 2.1
1.0 0.8 6.3 3.2 9.9 13.1 10.1 10.9 3.9 2.3 4.8 0.3 1.8 3.7 1.6 1.3
7.1
25.0
24
H. P. Baden
sults strongly support that hair contains a unique class of polypeptides. It is likely that they differ most from the epidermal proteins in the nonhelical regions, but this cannot be ascertained until the genes for the individual polypeptide are isolated and compared. The supramolecular structure of the fibrous proteins has not been established, but evidence for both the hair and epidermis indicates that the fundamental unit of the filament contains two helical polypeptides and that two such units (dimers) make up the most basic subunit. It has been clearly established that this basic subunit requires both acid and basic polypeptides and cannot be made from one alone.13y14 These dimers must then be organized by various types of packing into 80 A intermediate filaments. This packing could be parallel side to side and end to end, or some type of side-toside overlapping pattern. Finally, the fibrous proteins must be crosslinked to the matrix protein in which the filaments are embedded. This is likely carried out by copper or copper-containing enzymes. Presumably, this cross-linking occurs at the nonhelical ends of the fibrous proteins, which are rich in cysteine. Again, exact details of how this occurs are lacking, but the role of the matrix proteins in enhancing the tensile strength of hair is very clear, and this is presumably a result of the disulfide bonds that connect these two classes of proteins together. In addition to the cortical fibrous and matrix proteins, there are some other structural components of the hair that need to be considered. The major one is the cuticle layer, which really binds the cortical cells together. The cuticle cells, unlike the cortical cells, are surrounded by a cornified envelope that is highly resistant to mechanical injury.16 The outermost aspect of the cuticle cell contains a cystine-rich protein that provides mechanical strength. The cuticle cells contain only a small amount of fibrous protein that appears to be of the hair type (Franke W, personal communication). The need for the cuticle layer is apparent when one realizes that the cortical cells lack a cornified envelope, which is made up of cytoplasmic proteins cross-linked by transglutamase.16 Furthermore, the cortical cells lack
Clinics in Dermatology
lamellar bodies, so that material for holding the cells together is not secreted between the cells. The cortex differs from both the epidermis and nail by lacking these two components. The lack of lamellar bodies also results in the hair containing very little lipid (< 5%)so that it has poor barrier characteristics. This is also apparent in the capacity for hair to become rapidly hydrated and softened. This review has pointed out the complex chemical composition of hair. There are a number of cell types with different pathways of differentiation involving several molecular components. As our understanding of these evolves, we may learn how they may be related to disorders of hair growth and structure.
1. Fraser RDB, MacRae TP. Rogers GE. Keratins: Their compceition, structure and biosynthesis. Springfield, IL: Charles C Thomas, 1972;1-304. 2. Gillespie JM. The structural proteins of hair: isolation, characterization, and regulation of biosynthesis. In: Goldsmith LA, ed. Biochemistry and Physiology of the Skin, Vol. 1. New York: Oxford University Press, 1983:475-510. 3. Bereiter-Hahn J, Matoltay AG, Richards KS. Biology of the integument, II. Vertebrates. Berlin: Springer-Verlag, 19&1:293-317. 4. Hardy D, Bsden HP. Biochemical variation of hair keratins in man and non-human primates. Am J Phys Anthropol. 19X%39:19-24. 5. Jones LN. Studies on microfibrils from alpha-keratin. Biochim Biophys Acta. 1976;446:616-624. 6. Gillespie JM. The high-sulpher proteins of normal and aberrant keratins. In: Lyne AG, Short BF, eds. Biology of the Skin and Hair Growth. Sydney: Angus and Robertson, 1965377-398. 7. Crewther WG. Fraser RDB, Lennox FG, et al. The chemistry of keratins. Adv Protein Chem. 1965;20:191346. 8. Lee LD, Ludwig K, Baden HP. Matrix proteins of human hair as a tool for identification of individuals. Forensic Sci. 1978;11:115-122. 9. Marshall RC. Analysis of the proteins from single wool fibres by two-dimensional polyacrylamide gel electmphoreais. Text Res J. 1981;51:166-108. 10. Heid HW, Werner E, Franke WW. The complement of native alpha-keratin polypeptides of hair-forming cells: a subset of eight polypeptides that differ from epithelial cytokeratins. Differentiation. 1986;32:101-119. 11. Lynch MH, O’Guin WM, Hardy C, et al. Acidic and basic hair/nail (“hard”) keratins: their colocalization in upper
October-December 1988 Volume 6 Number 4
Biochemists of Hair Protein
cortical and cuticle cells of the human hair follicle and their relationship to “soft” keratins. J Cell Biology. 1986:103(6 Part 2):2593-2696. 12. O’Donnell IJ, Thompson EOP. Studies on reduced wool: the isolation of a major component. Aust J Biol Sci. 1964;17:973-989.
25
15. Hsshimoto K, Shibazaki S. Ultrastructural study on differentiation and function of hair. In: Kobori T, Montagna W, eds. Biology and Disease of the Hair. Tokyo: University of Tokyo Press, 197623-58.
13. &inert PM, Steven AC, Roop DR. The molecular biology of intermediate filaments. Cell. 1985;42:411-420.
16. Goldsmith LA. The epidermal cell periphery. In: Coldsmith LA, ed. Biochemistry and Physiology of the Skin, Vol. 1. New York: Oxford University Press, 1983;184196.
of the polypeptide 14. Lee LD, Baden HP. Organization chains in mammalian keratin. Nature. 1976;264:3’77379.
17. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24.
Address for correspondence: Howard P. Baden, MD, Harvard Medical School, Department Dermatology, Massachusetts General Hospital, Boston, MA 02114.
of
3 Howard P. Baden, MD
From the Department of Dermatology, Harvard Medical School, and Massachusetts General Hospital, Boston, Massachusetts
The hair shaft is surrounded by a layer of overlapping cuticle cells that are resistant to chemical and mechanical injury. These cells protect the cortex, which makes up the bulk of the hair shaft. The center of large hairs contains a medulla that is similar in composition to the inner root sheath but also has pigment granules. The fibrous character of hair results from the structural proteins in the cortical cell. Research on the chemistry of hair has been primarily directed toward studying these proteins. Various techniques have been used to isolate, purify, and characterize them.‘-3 The cortical proteins were found to be insoluble in neutral buffers and extractable with a minimum of degradation by using alkaline denaturing solvents containing a disulfide-reducing agent. Urea is a commonly used denaturingagent, and mercaptoethanol or dithiothreitol, or both, the reducing agents. In order to keep the proteins from aggregating, the cysteine residues were converted to S-carboxymethyl groups with iodoacetic acid. The protein solubilized from human hair is of two major classes: fibrous proteins that make up the filaments observed by electron microscopy and matrix proteins in which they are embedded.4-7 Electrophoretic and chromatographic studies show that both proteins contain a number of components that differ in size and chemical composition, although members of one class tend to be more similar to one another than to components from the other class. The major differences between the classes of proteins are their helical and cystine contents (Table 3-l). The fibrous protein had a helical content of 50% and contained 6 residues of )/zcystine per 100 amino acid residues, whereas the matrix proteins were nonhelical and had 25 to 30 residues of )/zcystine per 100 amino acid residues. An earlier technique used for separation was polyacrylamide gel electrophoresis in the presence of 6 M urea. This technique showed that the fibrous protein had four or more components, whereas the matrix proteins appeared as a single band (Fig. 3-l). When the electrophoresis was done at high gel densities, which is more effective in separating low-molecular weight proteins, a number of matrix proteins were visualized and the heterogeneity of the matrix proteins was better appreciated.8 The heterogeneity of the fibrous and matrix proteins was best demonstrated by two-dimensional electrophoresis using charge separation in the presence of urea in the first direction and size separation by SDS polyacrylamide gel electrophoresis in the second 22
October-December Volume 6 Number 4
1988 Biochemistrv
23
of Hair Protein
FIG. 3-1. Disc electrophoresis of the S-carboxymethyl hair proteins in 6 M urea. F is the fibrous polypeptides and M the matrix band. The arrow indicates the direction of electrophoresis.
direction.9 The matrix proteins proved to be even more complex when they were studied by chromatographic techniques. As a result of the work on the epidermal fibrous proteins, there was an interest in examining the fibrous proteins of hair in a more native state. The fibrous proteins exist in a reduced form in the lower hair shaft and become cross-linked by disulfide bonds when the cortical cells die and, of course, remain that way in the hair shaft. The major reason for studying the native proteins is that there are varying degrees of posttranslational modification of protein in cornified cells, and a clearer picture of the fibrous proteins could be obtained by studying them in their native reduced state. In order to isolate the native fibrous proteins of hair, the cortex of plucked hairs was dissected free of the inner and outer root sheaths. The tissue was homogenized, washed with water, and extracted either with a urea or SDS buffer. The protein extract was analyzed by two-dimensional electrophoresis, and basic and acid classes of fibrous polypeptides were found. There were four components in each, and they differed in mobility and size from the acidic and basic keratins of epidermis (Fig. 3-2).l’JBy isolating messenger RNA from the cortical cells, it was possible to show that they were all direct translational products and not derived one from the other by breakdown or posttranslational modification. Extraction of hair protein from the hair shafts without S-carboxymethylation followed by two-dimensional electrophoresis showed proteins similar to the native proteins described above,” but fewer individual components were identified and they differed somewhat in molecular weight and perhaps isoelectric points.
FIG. 3-2. Schematic diagram of two-dimensional electrophoresis of human epithelial (0) and hair (a) cytokeratins. A, P, and Bare standards. The numbers refer to the Moll17 catalog. (From Heid et al.,*0 with permission).
These proteins very likely correspond to the eight S-carboxymethyl polypeptides identified in wool, but direct comparison is not possible because of the modification induced by the Scarboxymethylation.12 Nevertheless, these reTABLE 3-1. Amino Acid Analyrls of the Alpha Fibrous (Alpha) and Matrix S-carboxymethyl Protolns of Halr. Data are Expressed as Residues per 100 ResiduesAmlno Adds
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine S-carboxymethyl cysteine
Alpha
Matrix
2.9 0.6 7.0 8.4 5.9 10.5 17.1 5.6 5.3 6.0 5.3 0.6 3.0 9.8 2.8 2.1
1.0 0.8 6.3 3.2 9.9 13.1 10.1 10.9 3.9 2.3 4.8 0.3 1.8 3.7 1.6 1.3
7.1
25.0
24
H. P. Baden
sults strongly support that hair contains a unique class of polypeptides. It is likely that they differ most from the epidermal proteins in the nonhelical regions, but this cannot be ascertained until the genes for the individual polypeptide are isolated and compared. The supramolecular structure of the fibrous proteins has not been established, but evidence for both the hair and epidermis indicates that the fundamental unit of the filament contains two helical polypeptides and that two such units (dimers) make up the most basic subunit. It has been clearly established that this basic subunit requires both acid and basic polypeptides and cannot be made from one alone.13y14 These dimers must then be organized by various types of packing into 80 A intermediate filaments. This packing could be parallel side to side and end to end, or some type of side-toside overlapping pattern. Finally, the fibrous proteins must be crosslinked to the matrix protein in which the filaments are embedded. This is likely carried out by copper or copper-containing enzymes. Presumably, this cross-linking occurs at the nonhelical ends of the fibrous proteins, which are rich in cysteine. Again, exact details of how this occurs are lacking, but the role of the matrix proteins in enhancing the tensile strength of hair is very clear, and this is presumably a result of the disulfide bonds that connect these two classes of proteins together. In addition to the cortical fibrous and matrix proteins, there are some other structural components of the hair that need to be considered. The major one is the cuticle layer, which really binds the cortical cells together. The cuticle cells, unlike the cortical cells, are surrounded by a cornified envelope that is highly resistant to mechanical injury.16 The outermost aspect of the cuticle cell contains a cystine-rich protein that provides mechanical strength. The cuticle cells contain only a small amount of fibrous protein that appears to be of the hair type (Franke W, personal communication). The need for the cuticle layer is apparent when one realizes that the cortical cells lack a cornified envelope, which is made up of cytoplasmic proteins cross-linked by transglutamase.16 Furthermore, the cortical cells lack
Clinics in Dermatology
lamellar bodies, so that material for holding the cells together is not secreted between the cells. The cortex differs from both the epidermis and nail by lacking these two components. The lack of lamellar bodies also results in the hair containing very little lipid (< 5%)so that it has poor barrier characteristics. This is also apparent in the capacity for hair to become rapidly hydrated and softened. This review has pointed out the complex chemical composition of hair. There are a number of cell types with different pathways of differentiation involving several molecular components. As our understanding of these evolves, we may learn how they may be related to disorders of hair growth and structure.
1. Fraser RDB, MacRae TP. Rogers GE. Keratins: Their compceition, structure and biosynthesis. Springfield, IL: Charles C Thomas, 1972;1-304. 2. Gillespie JM. The structural proteins of hair: isolation, characterization, and regulation of biosynthesis. In: Goldsmith LA, ed. Biochemistry and Physiology of the Skin, Vol. 1. New York: Oxford University Press, 1983:475-510. 3. Bereiter-Hahn J, Matoltay AG, Richards KS. Biology of the integument, II. Vertebrates. Berlin: Springer-Verlag, 19&1:293-317. 4. Hardy D, Bsden HP. Biochemical variation of hair keratins in man and non-human primates. Am J Phys Anthropol. 19X%39:19-24. 5. Jones LN. Studies on microfibrils from alpha-keratin. Biochim Biophys Acta. 1976;446:616-624. 6. Gillespie JM. The high-sulpher proteins of normal and aberrant keratins. In: Lyne AG, Short BF, eds. Biology of the Skin and Hair Growth. Sydney: Angus and Robertson, 1965377-398. 7. Crewther WG. Fraser RDB, Lennox FG, et al. The chemistry of keratins. Adv Protein Chem. 1965;20:191346. 8. Lee LD, Ludwig K, Baden HP. Matrix proteins of human hair as a tool for identification of individuals. Forensic Sci. 1978;11:115-122. 9. Marshall RC. Analysis of the proteins from single wool fibres by two-dimensional polyacrylamide gel electmphoreais. Text Res J. 1981;51:166-108. 10. Heid HW, Werner E, Franke WW. The complement of native alpha-keratin polypeptides of hair-forming cells: a subset of eight polypeptides that differ from epithelial cytokeratins. Differentiation. 1986;32:101-119. 11. Lynch MH, O’Guin WM, Hardy C, et al. Acidic and basic hair/nail (“hard”) keratins: their colocalization in upper
October-December 1988 Volume 6 Number 4
Biochemists of Hair Protein
cortical and cuticle cells of the human hair follicle and their relationship to “soft” keratins. J Cell Biology. 1986:103(6 Part 2):2593-2696. 12. O’Donnell IJ, Thompson EOP. Studies on reduced wool: the isolation of a major component. Aust J Biol Sci. 1964;17:973-989.
25
15. Hsshimoto K, Shibazaki S. Ultrastructural study on differentiation and function of hair. In: Kobori T, Montagna W, eds. Biology and Disease of the Hair. Tokyo: University of Tokyo Press, 197623-58.
13. &inert PM, Steven AC, Roop DR. The molecular biology of intermediate filaments. Cell. 1985;42:411-420.
16. Goldsmith LA. The epidermal cell periphery. In: Coldsmith LA, ed. Biochemistry and Physiology of the Skin, Vol. 1. New York: Oxford University Press, 1983;184196.
of the polypeptide 14. Lee LD, Baden HP. Organization chains in mammalian keratin. Nature. 1976;264:3’77379.
17. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24.
Address for correspondence: Howard P. Baden, MD, Harvard Medical School, Department Dermatology, Massachusetts General Hospital, Boston, MA 02114.
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