Biophysical Characterization of Involucrin Reveals a Molecule Ideally ...

Vol . 267, No. 17. Issue of June 15, pp,12233-12238,1992 Printed in U.S. A.

CHEMISTRY THEJOURNAL OF BIOLOGICAL 8 1992 by The American Society for Biochemistry and Molecular Biology,, Inc

Biophysical Characterizationof Involucrin Reveals a Molecule Ideally Suited to Functionas an Intermolecular Cross-bridgeof the Keratinocyte Cornified Envelope* (Received for publication, September 30, 1991)

Michael B. YaffeSO, Helga Beegenll, and Richard L. Eckert$llll**$$ From the Departments of $Physiology and Biophysics, IIDermatology, **Reproductiue Biology and VBiochemistry, Case Western Reserue University School of Medicine, Cleueland, Ohio 44106

Involucrin is a 68-kDa precursor of thekeratinocyte cornified envelope. During keratinocyte terminal differentiation glutamine residues of involucrin become covalently cross-linked to other envelope precursors via covalent e-(y-glutamy1)lysine bonds. In the present study we examine the secondary andtertiary structure of human involucrin using computer algorithms, circular dichroism, and electron microscopy.Our results indicate that involucrin is an extended, fleFible, rodshaped molecule that has a length of 460 A, an axial ratio of 30:l and possesses between 60 and 75% ahelical content. Glutamine residues are circumferentially distributed along the length of the a-helical segis conserved ments of the molecule, a distribution that in all species. We hypothesize that this distribution of glutamine residues togetherwith the elongated shape of the molecule permits optimal interaction ofinvolucrin glutamyl side chains with the lysine residues of other para-membranous proteins during transglutaminase-mediated cross-linking. Moreover, its long length allows involucrin to cross-link molecules that are separated by substantial distances in the cornified envelope. These properties allow a single involucrin molecule to form multiplecross-links, in multiple spatial planes, with other envelope precursors. Thus, the structure of involucrinis that of an ideal intermolecular cross-bridge.

The cornified envelope is the terminal product of keratinocyte differentiation (Matoltsy and Matoltsy, 1966; Green, 1979; Rice and Green, 1979). The envelope forms adjacent to the cytoplasmic face of the plasma membrane througha protein-protein cross-linking reaction catalyzed by keratinocytetransglutaminase,acalcium-dependent(Buxman and Wuepper, 1976;Ogawa and Goldsmith, 1976), membranebound enzyme (Thacher and Rice, 1985). The cross-links consist of covalent c-(y-glutamy1)lysinebonds (Green, 1979). Involucrin, one of the precursors of the cornified envelope, is a soluble cytosolic protein that serves as themajor glutamyl donor in the transglutaminase-catalyzed cross-linking reaction anddisappears from the soluble phase into theparticulate

* This work was supported by Grant GM43751 from the National Institutes of Health and Grant AR39750 from the Skin Diseases Research Center of Northeast Ohio. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by NationalInstitutes of HealthTrainingGrant HL07653. $$ To whom correspondence should be addressed. Tel.: 216-3685530.

fraction following calcium-dependent activation of transglutaminase (Rice and Green, 1979; Simon and Green, 1984, 1985, 1988; Rorke and Eckert, 1991). The involucrin amino acid sequence, determined from the cloned gene (Eckert and Green, 1986), contains 25 mol % glutamine and 20 mol % glutamate. A striking feature of the molecule is the presence of a central segment composed of39 tandem repeats of 10 amino acids each (Eckertand Green, 1986) (Fig. 1). The central segment is flanked by 153 amino acid N-terminal and 45 amino acid C-terminal segments that lack the repeat structure (Eckert andGreen, 1986). The consensus sequence of thecentral segment repeats is (ELP/KHL)EQQEGQL (Eckert and Green, 1986). The repeating structure of the central segment is conserved in involucrins from all higher primates,although the number of tandemrepeats varies (Tseng andGreen, 1988). This arrangement of glutamine-rich repeating elements is also found in lower primates and in the pig, although here the repeat length has been increased to 13 and 16 amino acids, respectively (Tseng and Green, 1988, 1990; Phillips et al., 1990). In spite of considerable progress indemonstrating that involucrin is a specific substrate for transglutaminase (Rice and Green, 1979; Simon and Green, 1984, 1985,1988; Rorke and Eckert, 1991), the secondary and tertiary structure of involucrin has not been thoroughly studied. Thus, whether involucrin is a rod-shaped or globular molecule and whether the glutamine residues are clustered or dispersed on the surface influence how it may function as a cornified envelope constituent and substrate for transglutaminase. In the absence of this knowledge, it is difficult tounderstandthe molecular mechanisms by which involucrin functions as an envelope precursor. In the present study, we use circular dichroism and electron microscopy to show that human involucrin is a long, thin, flexible rod-like molecule containing a significant amount of a-helical content. Computer-based analysis coupled with experimental measurement of secondary and tertiary structuresuggests that the molecular shape and thedistribution of glutamine residues along the involucrin molecule are important for its role as anintermolecular crossbridge. We present a model of the molecule that is consistent with our physical measurements and with a role for involucrin as a major molecular cross-bridge of the cornified envelope. EXPERIMENTALPROCEDURES

Keratinocyte Cell Culture-Human foreskinkeratinocytes were cultured as described by Rheinwald and Green (1975) using a 3:l mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium supplemented with 8% fetal calf serum and other additives (Eckert and Green, 1984). At confluence, the concentration of fetal calf serum was reduced to 5%, and epidermal growth factor was omitted to promotedifferentiation (Sun and Green, 1976). Some

12233

12234

Biophysical Properties of Human Involucrin

- -b

CENTRAL SEGMENT c m

FIG. 1. Computer prediction of involucrin secondary structure and hydrophobicity. The top panel shows a scale drawing of the involucrin primary sequence. The central segment is composed of39 contiguous 10 amino acid repeats (open rectangles). The N-terminaland C-terminal-flanking segments are less highly conserved and comprise 153 and 45 amino acids, respectively. Computer analysis of involucrin secondary structure (Garnier et al., 1978), shown in the center panel, predicts that most of the central segment is a-helical (open rectangles). The helical regions are separated by short random coil segments (solid rectangles). Three @-strandregions are predicted near the amino terminus and four @-strandregions near the carboxyl terminus (slashed rectangles). Predictions of reverse turn (hatched rectangles) are rare, with one such region near the N terminus, and another near the C terminus. However, this latter region has nearly equal propensity to form a random coil. The hydrophobicity profile (Kyte and Doolittle, 1982) is shown in the bottom panel. Hydrophilic regions are shown below and hydrophobic regions above the dashed line. The profile reveals that the most of the molecule is hydrophilic, with the exception of several discrete hydrophobic domains in the vicinity of the predicted @-strands at the N and C terminus. cultures were labeled with 250 pCi of [35S]methioninefor 16-20 h prior to harvest (Gorodeski et al., 1989). Znuolucrin Purification-Involucrin was purified from cultured human keratinocytes by heat fractionation and anion-exchange high pressure liquid chromatography (HPLC).' Nineteen 50-cm2dishes of human foreskin keratinocytes, and one plate of [35S]methioninelabeled keratinocytes were washed twice with phosphate-buffered saline containing 20 mM EDTA, once with phosphate-buffered saline containing 20 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride, and harvested by scraping. Crude cell homogenates were prepared by two freeze-thaw cycles followed by Dounce homogenization at 4 "C. After centrifugation at 100,000 X g for 30 min at 4 "C, the supernatant was supplemented to 62.5 mM Tris-HC1, pH 6.8, and 10% glycerol, and heat fractionated at 95 "C for 10 min as previously described (Simon and Green, 1985; Etoh et al., 1986). Samples were cooled on ice for 15 min, centrifuged a t 100,000 X g (4 "C) for 30 min, and the involucrin-enriched supernatant (-4 ml) was applied to a Mono-Q anion-exchange HPLC column (Pharmacia LKBBiotechnology Inc.). Proteins were eluted using a 40-min linear gradient of 0-1.0 M NaCl in 20 mM sodium phosphate, pH 6.8. at a flow rate of 1 ml/min. Fractions (500 pl) were collected, and 50 p1 was removed for scintillation counting. Fractions corresponding to peaks of radiolabel were pooled, dialyzed overnight against two changes of 1 mM ammonium bicarbonate containing 0.2 mM phenymethylsulfonyl fluoride, lyopholized, and stored at -20 "C. In some experiments, Mono-Q-purified involucrin was further purified by chromatography on a 1.6 X 56-cm Bio-Gel A1.5M column developed isocratically a t 4 "C in phosphate-buffered saline containing 0.02% sodium azide at a flow rate of 0.5 ml/min. Under these conditions involucrin elutes near the void volume (Rice and Green, 1979). Circulur Dichroism Spectroscopy-Circular dichroism (CD) spectra were measured using a computer-controlled Jasco 5600 spectrometer calibrated with (+)-IO-camphor sulfonic acid (Chen and Yang, 1977). Spectra were scanned at 50 nm/min, digitized, and recorded at 0.2nm intervals. All spectra were measured at room temperature using a 0.45-cm path length cuvette and averaged over six individual scans. Electron Microscopy-Involucrin was negatively stained for electron microscopy using a procedure modified from Green et al., 1972. Involucrin samples were diluted to 100 pg/ml in 20 mM Tris-HC1, pH 7.0, and adsorbed beneath a carbon film coated on freshly cleaved mica. The carbon films were floated off the mica in solutions containing either 1%sodium silicotungstate, pH 7.0, 1% ammonium The abbreviations used are: HPLC, high performance liquid chromatography; SST, sodium silicotungstate.

molybdate, pH 7.2, or 0.5% uranyl acetate, pH 4.8, picked up on copper grids and examined using a JEOL-100C electron microscope at a nominal magnification of X60,OOO. The microscope wascalibrated using the 87.5 8, spacing of catalase crystals (Wrigley, 1968). Involucrin molecular dimensions were measured to thenearest 0.5 mm from photomicrographs prepared at a magnification of X147,OOO. Gel Electrophoresis and Zmmunoblotting-Samples were electrophoresed using 6.5% polyacrylamide denaturing gels (Laemmli, 1970), transferred to nitrocellulose, and incubated with a polyclonal rabbit anti-involucrin antibody (Rice and Green, 1979) as described previously (Gorodeski et al., 1989). Protein concentrations were determinedusing the bicinchoninic acid assay procedure with bovine serum albumin as the standard. The amino acid concentration of purified involucrin samples used for CD measurements was determined by quantitative amino acid analysis (Mays and Rosenberry, 1981). ComputerAnalysis-Computer analysis of secondary structure and hydrophobicity was performed using the Intelligenetics PCGENE software package. RESULTS

Computer Modeling Predicts That Involucrin Is Hydrophilic and a-Helical-Computer analysis of secondary structure (Garnier et al., 1978) predicts that 75% of the involucrin molecule, including most of the central segment, is strongly a-helical (Fig. 1, center panel). The a-helical segments (open rectangles) are separated by short segments of random coil in the vicinity of proline residues (dark rectangles). In addition, most of the a-helical portions of the molecule are predicted to be hydrophilic (Fig. 1,lowerpanel). In contrast, the extreme amino and carboxyl termini arepredicted to contain, respectively, three and four relatively hydrophobic p-strands (Fig. 1, center and lower panels). Interestingly, there are few predicted reverse turns (Fig. 1, center panel, cross-hatched rectangles); one is present just before the third predicted pstrand at the extreme N terminus, and a second is present just before the third p-strand in the extreme C terminus. However, the latter p-turnis predicted to have a nearly equal propensity to form a random coil (solid and cross-hutched rectangles). Involucrin Purification-Involucrin was purified from crude keratinocyte homogenates (Fig. 2). Progress of the purifica-

Biophysical Properties A

Fraction Number

1 180-

2

3

4

B

a k

116-

c

845848

-

33-

1 180-

2

3

4

c

-

D

116-

84-

48

-

36

-

FIG.2. Involucrin purification. Keratinocyte homogenates were centrifuged to yield a “crude” supernatant which was then heated and recentrifuged (“ExperimentalProcedures”).The resulting supernatant was applied to a Mono-Q HPLCcolumn (panel A ) and eluted using a linear sodium chloride gradient (dashed l i n e ) . Radiolabel eluted as two peaks at 0.41 (I)and 0.50 M NaCl (II) ( t o p panel). The purification process was monitored by 6.5% polyacrylamide gel electrophoresis of samples (approximately 5 pg of protein) a t each step. Panel R shows a Coomassie-stained gel of crude supernatant (lane I ), heat-treated supernatant (lane 2), and peaks I (lane 3 ) and I1 (lane 4 ) from the Mono-Q column. Panel C shows the fluorogram of the same gel. In panel I), protein from peak I from the Mono-Q column was electrophoresed,transferred to nitrocellulose,and incubated with anti-involucrin antibody (Rice and Green, 1979). The arrows in panels R-D indicate the migration of involucrin. The band at 58 kDa in lane D is a n involucrin breakdown product that represents 90% involucrin (Fig. 2, B and C , lane 3 ) ;peak I1 elutes at 0.50 M NaCl and contains FIG.3. Circular dichroism spectra of involucrin indicate a higher and lower molecular weight material (Fig. 2, B and C, high u-helical content. .Mono-()-purified involucrin W R R further lane 4 ) . As expected, the Mono-Q-purified material migrates purified hy Hio-Gel A1.5M size exclusion chromatography. dialyzed at approximately 90 kDaonadenaturingacrylamide gel against 1 mM ammonium bicarbonate. and Ivopholized. The sample was reconstituted in 20 mM sodium phosphate. pH 7 . 0 . and its circular (Simon and Green, 1984, 1985) and reacts with a polyclonal dichroism spectrum was measured using a .Jasco .J6OO spectrometer antibody specific for human involucrin (Fig. 2 0 ) . We rou(panel A ) . Involucrin samples, reconstituted as in pond A , were tinely obtained 2400 pg of Mono-Q-purified involucrin from acldified by addition of HCI and the CD spectra recorded ( p o n d I { , twenty 50-cm2 dishes of 10 day post-confluent keratinocytes. trace 1, pH 7.0; trace 2. pH 6.0; rraw 3 . pH 3.0; frncr 4 . pH 2 . 0 ) .

-

12236

Biophysical Properties of Human Involucrin

Optimal resolution was obtained using sodium silicotungstate the three and four amino acid spacings between glutamate (SST), although similarimages were obtained using eitherof and lysine residues within the 10 amino acid repeat allows the other negative stains (not shown). Involucrin molecules ion-pair interactions between these residue's side chains, furnegatively stained with SST appearas long thinrods of therstabilizingthen-helicalconformation(Marquseeand Baldwin, 1987). An additional13% of the molecule is prerelatively uniform length (Fig. 4). These rods must possess some degree of flexibility since many of the molecules are dictedto bein therandom coil conformation,duetothe bent, curved, or kinked a t various positions. We believe that disruptiveinfluence of proline residues at variouspoints within the helical segment (Woolfson and Williams, 1990). each rod in the SST preparationcorrespondstoasingle Direct measurements of secondary structure using circular involucrin molecule (see below and "Discussion"). T o determine the dimensions of the involucrin molecule, dichroism revealed that at neutral pH approximately 50% of the contour lengths of 165 rods in SST-stained preparations the molecule is n-helical and approximately 40% is random of involucrin was approxweremeasured (Fig. 4, rightpanel). Thedistribution of coil. The measured 8-sheet content molecular lengths was r o y h l y gaussian and revealed a mea? imately 1076, in good agreement with the 8% predicted by molecular length of 460 A with a standard deviation of 70 A. computer analysis (Fig. 1). Thus, the computer-hased strucNone of the measured lengths exceed the theoretical contour ture predictions arein good agreement with the experimental length expected if involucrin were a completely n-helical rod measurements, exceptfor a slight discrepancyin the estimate (880 A), consistent with each rod corresponding to a single of n-helical content. This is not unexpected since secondary molecule. structurepredictionalgorithms may over-estimatethe nhelical content of glutamicacid-rich molecules (Nagasawa DISCUSSION and Holtzer, 1964; Blank et al., 1986). while CD spectroscopy may underestimate helical content as a result of minor disInvolucrin is an important precursor of the keratinocyte cornified envelope and is a major substratefor transglutamin- tortions within the helix (Manning et al., 1988). Electron microscopic observation of negatively stained inase (Rice and Green, 1979; SimonandGreen, 1984, 1985, 1988; Rorke and Eckert, 1991). T o understand the role of volucrin preparations revea!ed thin, flexible rodshaving a involucrin as an envelope constituent and as a substrate of mean contour lengthof 460 A. If 75% of the molecule were crhelical, as suggested by the computer-based algorithms (Fig. transglutaminase, we have usedphysical methods to learn l), and the helical segments were arranged e?d-to-end, the more about its structure. Computer-based secondary structure predictionssuggested helical portion of the molecule would be 660 A in length. If that 75% of the involucrin molecule, including most of the 50% of the molecule were n-helical, as estimatedhy CD, and central segment, is n-helical (Fig. 1).This large helical pro- the helical segments were contiguous, the helical portion of pensity is a consequence of the high proportion of helix- the molecule would be 440 A in length. For comparison, a stabilizing residues within the 10 amino acid repeating ele- globular molecule having the samemolecular weight as involments, notably leucine, glutamate, and glutamine (Fig. 5 R ) ucrin would have a radius of gyration of only 30 A assuming a specific volume of 0.74 cm:'/g (Cantor and Schimmel,1980). (Gamier et al., 1978; Chou and Fasman, 1974). In addition,

FIG. 4. Electron microscopy of involucrin molecules rrvrals long:, thin rods. Mono-Q-purified involucrin in 20 mM Tris-HCI. pH 6.8 (approximately 200 pg protein/rnl) was negatively stained with sotliurn silirotungstate and examined hv electron microscopy. The riphr panel shows a histogram o f measured contour lengths for 165 individual involucrin molecules. The mean contour length was me.mured at , $ I N 2 70 A. The average persistence length (the straight line dist,ance from one end of the rod to the other) was measurrd as 340 A 174p; of the mean contour length), a value that is consistent with involucrin heing an extended molecule.

12237

Biophysical Properties of Human Involucrin

A

11

lnvoiucrin

?

10 Amlno Acid

Consensus Repeat

2

1

3

”3 1 . 15

11 Amlno Add Consensus Repeat

19Tums

19Turns

I.

FIG. 5. A model for the structure of h u m a n involucrin. Our results suggest thnt involucrin has a central region dominated hy ( t helical segments (open rectang1c.s) interspersed with short relatively flexible regions of random coil (solid lincs). The N - and r-terminal segments are modeled as hydrophohic &sheet structures (panel A ) . 1’anc.l H demonstrates the effect of changing the lengh ofthe repeats in the central rod domain. The helical wheel presentation of the 10-amino-acid involucrin consensus sequence (uppvr srqurncc. and lr/f whw1. panel H ) shows the uniform circumferential distrihution of glutamine residues that results from 19 turns of the involucrin n-helix. In contrast. an art,ificial 1 1 amino acid repeat generated by inserting an extra amino acid ( X ) hetween the consensus 1 0 amino acid repeats results in a clustering.. of ,. glutamine residues on one face of the helix (lower sewence and right w h e d , pond HI. The numhrrs indicate the order of the glutamine residues as one proceeds along the helix.

Therefore,ourmeasuredcontourlength (460 A) ismost diameter give an axial ratio of3O:1, in excellent agreement consistent with a long and extended linearmolecule. with the 3O:l axial ratio previously reported for involucrin on The diameterof the rods couldnot be determined withgood the basis of its measured hydrodynamic properties (Rice and reliability, andwere therefore estimated using theformula: d Green, 1979). Finally, examinationof the involucrin sequence reveals no evidence for a heptad repeat structure character= 2 [M,W/(LTNA)]’/’ (Odermatt et af., 1982). This formula assumes that themolecule has a cylindrical shape, where M , istic of proteins that form coiled-coil dimers (McLachlan and is the molecular weight, L is the measured contour length, w Karn, 1983), andi t is well established that a-helical peptides isthe specificvolume (0.74 cm’/g) and NA is Avagadro’s can exist in solution as stahle monomers (Marqusee et al., number. The diameterof the involucrin molecule, calculated 1989; Kim and Raldwin, 1984; Bradley et af., 1990). in this way, is 15 A. A model for the structure of involucrin based on the preThereare several lines of evidence suggesting that the dicted secondary structure, CD spectra. and EM images is involucrin molecules we observe in SST-stained preparations shown in Fig. 5A. In thismodel, most of the molecule is shown are monomers. First, involucrin migrates as a single band in to consist of segments of a-helix (rwtangukar h o x ~ s )joined , non-denaturing polyacrylamide gels (Etoh et al., 1986) and its a t various angles by flexible segments of random coil (solid mobility is identical whether the sample is boiled in detergent lines). The N and C termini are modeled as hydrophobic dor maintained in its native condition prior to electrophoresis sheets, as predicted in Fig. l. The position of glutamine 496, a residue previously shown (Etoh et al., 1986). Second, involucrin sediments as a single to be highly reactive in vitro, is indicated by a closed square, band in sedimentationequilibriumexperiments(Riceand Green, 1979). Third, a molecular mass of 83 kDa is estimated whereas the positionsof other moderately reactive glutamine using sedimentation and diffusion coefficients and the Sved- residues are indicated by open symbols (Simon and Green, berg equation (Rice and Green, 1979). This is consistent with 1988). The region of the molecule indicated in Fig. 5A by an the known molecular mass of an involucrin monomer, 68 kDa,asterisk encompasses a particularly non-reactive setof glutaderived from the gene sequence (Eckert and Green, 1986) and mine residues (Simon and Green, 1988). It is interesting to speculate that this low reactivity may result from the fact is not consistent with doublet (136 kDa) formation. Fourth, the molecules visualized in our electron micrographs display that a significant fraction of this segment is predicted to be a monodisperse distribution of contour lengths suggestive of in the random coil conformation (Fig. 1). In fact, the confora single species; moreover, we observed no evidence of molec- mation of the region may account for the difference between the 50% a-helical content estimated by CD and the ?Fir; a ular “fraying” or breakdown into smaller subunits (Fig. 4). Fifth, our measured contour length and calculatedmolecular helical content predicted by the computer-based algorithm

12238

Biophysical Propertiesof Human Involucrin

(i.e.a larger fraction of this segment may berandom coil than indicated in the model). The high a-helical content of involucrin has important implications for the distribution of glutamine residues along the molecule. Fig. 5B (top left) shows a helical wheel representation (Schiffer and Edmundson, 1967)for involucrin composed of repeats of the involucrin consensus sequence, KHLEQQEGQL (Eckert and Green, 1986). The linear sequence is shown at the bottom with the glutamine residues indicated by cross-hatched boxes. This represents 19 turns around the a-helix (total of 68 residues). The relative position of each glutamine residue (cross-hatched oual) on the helical wheel are numbered as one progresses down the axis of the a-helix. It is evident that thecombination of a 10-amino-acid repeating element and an a-helical conformation results in the uniform circumferential distribution of glutamines along the length of the helix. Our analysis reveals a similar circumferential distribution of glutamine residues for the 13-aminoacid repeat found in the galago and lemur (Tseng and Green, 1988; Phillips et al., 1990), and for the 16 amino acid repeat found in the pig (Tseng and Green, 1990) (data not shown), suggesting that thisdistribution may have some evolutionary significance. In contrast,if the length of the repeating unit is artificially increased to 11 residues by adding an additional amino acid, X , to each repeat (Fig. 5B, bottom),the glutamine residues (dark ouak) cluster ononly one face of the helix (Fig. 5B, top right). Since each glutamineresidue is a potential site of transglutaminase-mediated cross-linking, this constricted distribution might render involucrin less effective in forming protein-protein cross-links (for example by steric hindrance between cross-linked molecules clustered on one helical face). If the repeat length is reduced to nineamino acids, KHLEQQEGQ, the glutamine residues are distributed around the a-helix, but are restricted to only six of the possible 18 positions and therefore project into fewer spatial planes (not shown). Thus, the combination of an a-helix and an evolutionarily conserved 10-amino-acid repeat length, assuresthat glutamine residues will be circumferentially distributed in all positions around the helix. This glutamine distribution coupled with the long extended nature of the molecule maximizes the ability of involucrin to form cross-links in all spatial planes and to cross-link molecules separated by substantial distances in the envelope. During keratinocyte maturation and migration from the basal layer to the body surface, the intracellular pH becomes increasingly acidic. We therefore measured the CD spectra of involucrin under conditions where the pH was varied from 7 to 2. Even at pH 2, involucrin retains considerable (approximately 40%) a-helical content (Fig. 3B). Thus, involucrin retains its functional conformation even at intracellular pH values far below those expected in terminally differentiated keratinocytes. In summary, our results indicate that the glutamyl donor sites on involucrin are distributed circumferentially along an a-helical rod due to the maintenance of a defined repeat length. While the repeat length varies in differentspecies, the circumferential distributionof glutamine residues is retained. We hypothesize that this distribution permits optimal interaction of involucrin glutamyl side chainswith the lysine

residues of para-membranous proteins during transglutaminase-mediated cross-linking, allowing a single involucrin molecule to form multiple cross-links with other envelope precursor proteins. Several differentprecursors of the cornified envelope have been identified, and some of them, such as loricrin, may comprise a significant proportion of the envelope (Mehrel et al., 1990). Our model suggests that involucrin could facilitate cross-link formation, without necessarily constituting the major component of the envelope by mass. In this respect, involucrin is ideally suited to function as an intermolecular cross-bridge of the cornified envelope. Acknowledgments-We gratefully acknowledge the contribution of

Dr. Terry Rosenberry(Department of Pharmacology, Case Western) for performingthe amino acid analysis and Dr. Gary Bright (Departments of Physiology and Biophysics, Case Western) for discussions regarding contour lengths. REFERENCES Alder, A. J., Greenfield, N. J., and Fasman,G. D. (1973) Methods Enzymol. 2 7 , 675-735 Blank, G. S., Yaffe,M.B., Szasz, J., George, E., Rosenberry, T. L., and Sternlicht, H. (1986) Ann. N. Y. Acad. Sci. 466,467-481 Bradley, E. K., Thomason, J. F., Cohen, F. E., Kosen, P. A., and Kuntz, I. D. (1990) J.Mol. Biol. 216,607-622 Buxman, M. M., and Wuepper, K. D. (1976) Biochim. Biophys. Acta 452,356369 Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, p. 554, W. H. Freeman and Company, San Francisco Chang, T. C., Wu, C.-S. C., and Yang, J. T. (1978) Anal. Biochem. 9 1 , 13-31 Chen, G. C., and Yang, J. T. (1977) Anal. Lett. 10,1195-1207 Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13,211-222 Eckert, R. L., and Green, H. (1984) Proc. Natl. A c d . Sci. U. S. A. 8 1 , 43214325 Eckert, R. L., and Green, H. (1986) Cell 46,583-589 Etoh, Y., Simon, M., and Green, H. (1986) Biochem. Biophys. Res. Commun. 136,51-56 Garnier J., Os thorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120,97-120 Gorodeiki, G. Eckert, R. L., Utian, W. H., and Rorke, E. A. (1989) Endocrinology 126,399-406 Green, H. (1979) Harvey Lect. 7 4 , 101-139 Green, N. M., Valentine, R. C., Wrigley, N. G., Ahmad, F., Jacobson, B., and Wood, H. G. (1972) J.Bid. Chem. 247,6284-6298 Hennessey, J. P., Jr., and Johnson, W. C., Jr. (1981) Biochemistry 2 0 , 10851094 Holzwarth, G., and Doty, P. (1965) J.Am. Chem. SQC.87,218-228 Kim, P. S., and Baldwin, R. L. (1984) Nature 307,329-334 Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132 Laemmli, U. K. (1970) Nature 227,680-685 Manning, M. C., Illangasekare, M., and Woody, R. W. (1988) Biophys. Chem. 3 1.77-86 Marqusee, S., and Baldwin, R. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 8898-8902 Marqusee, S., Robbins, V. H., and Baldwin, R. L. (1989) P m . Natl. A d . Sci. U. S. A. 86,5286-5290 Matoltsy, A. G., Matoltsy, M. N. (1966) J.Inuest. Dermatol46, 127-129 Mays, C., and Rosenberry, T. L. (1981) Biochemistry 20,2810-2817 McLachlan, A. D., and Karn, J. (1983) J. Mol. Biol. 164,605-626 Mehrel, T., Hohl, D., Rothnagel, J. A., Longley, M. A., Bundman, D., Cheng, C., Lichti, U., Bisher, M. E., Steven, A. C., Steinert, P. M., Yuspa, S. H., and Roop, D. R. (1990) Cell 6 1 , 1103-1112 Nagasawa, M., and Holtzer, A. (1964) J. Am. Chem. SOC.86,538-543 Odermatt, E., Engel, J., Richter, H., and Hormann,H. (1982) J. Mol. Biol. 1 6 9 , 109-123 Ogawa H. and Goldsmith L. A. (1976) J . Biol. Chem. 2 6 1 , 7281-7288 Phillip's hi. Di'an P. and Green, H. (1990) J. Biol. Chem. 266,7804-7807 Rheinwhd, >. dnd'Green, H. (1975) Cell 6 , 331-344 Rice, R. H., and Green, H. (1979) Cell 18,681-694 Rorke, E. A., and Eckert, R. L. (1991) J.Inuest. Dermatol. 97,543-548 Saxena, V. P., and Wetlaufer, D. B. (1971) Proc. Natl. Acad. Sci. U. S. A . 6 8 ,

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