The proteins of the embryonic chick epidermis : I. During the normal development in ovo

As a preliminary to a study of the proteins of the embryonic chick epidermis when grown in vitro under various culture conditions, the proteins of the anterior metatarsal epidermis, from 11 days of embryonic life up to 9 days posthatching, have been studied. Carboxymethylated derivatives of the proteins extracted by a thiol reduction procedure have been analyzed by polyacrylamide gel electrophoresis. The results have shown that the differentiation of the epidermis is characterized by the appearance between days 14 and 17 of at least 11 major protein bands in the electrophoretic pattern. Two of these bands are of relatively high molecular weight protein and appear earlier than the remaining bands which form a group of closely related, low molecular weight protein species. The differentiation of the tissue also involves the disappearance from the electrophoretic pattern of all but one of the five major bands present in extracts of the 11/12-day epidermis. A study of the proteins derived from the isolated periderm of the 14-day chick embryo beak has suggested that one of the major bands in the 11/12-day metatarsal epidermal extracts may be a peridermal protein.     

The proteins of living psoriatic epidermis

Psoriatic epidermis has a rapid rate of tunrover and produces a stratum corneum with an abnormal tonofilament composition. One polypeptide chain, (M_r 70 000) is absent or greatly decreased in relative amount and two other chains, (M_r 63 000 and 55 000), which are normally modified in the living cells, persists into the stratum corneum. Increasing the turnover of normal epidermis has been shown to cause the persistence of 63 and 55 kilodalton chains in the stratum corneum but does not affect the relative amount of 70 kilodalton chain. It has, therefore, been suggested that, in psoriasis, the deficiency of the 70 kilodalton chain may occur prior to or simultaneuously with the induction of increased tissue turnover. In the present study, the polypeptide chain composition of living psoriatic epidermis has been examined. It is shown that the relative amounts of 70 kilodalton chain in psoriatic stratum corneum and involved living epidermis from the same site are not significantly different. The abnormality is therefore, already present in the living cells and it appears that, in psoriasis, the synthesis of the 70 kilodalton chain is defective. The uninvolved epidermis of psoriatics is intermediate between normal and involved psoriatic epidermis both in the ability to synthesise the 70 kilodalton chain and to modify the 63 and 55 kilodalton chains. Comparisons of amino acid compositions of proteins containing different proportions of 70 kilodalton chain suggest that it has a considerably higher content of glycine and serine than the other tonofilament chains. These studies suggest that the 70 kilodalton chain may be functionally different from the other tonofilament chains. The defect in its synthesis in psoriasis is a relatively early event and may be involved with the induction of increased tissue turnover or induced by the same abnormal conditions as the increased tissue turnover.     

The proteins of the embryonic chick epidermis : II. During culture in serum-containing medium with and without added vitamin A

The proteins of the 12-day embryonic chick anterior metatarsal epidermis have been studied during growth in vitro in a serum containing medium with and without added vitamin A (5 IU/ml). The keratinization observed in the serum-containing medium alone was thus shown to be defective since only two of the proteins associated with keratinization during development in ovo were synthesized by the cultured epidermis, whereas the major group of 9 keratin protein bands was almost completely absent. The possible structural origins of these keratin protein bands is discussed in the light of these findings.In the medium containing vitamin A, synthesis of the two keratin proteins observed in the control epidermis was prevented and instead the band pattern obtained from the retinol-treated epithelium remained very similar to that of the 12-day epidermal starting material. Certain bands were increased in intensity in the presence of vitamin A, however, and in particular, the major band of the 12-day epidermis, which appears to be peridermal in origin, was present in increased amounts.     

Region-specific deposition of dermal proteins between dermis and epidermis during induction of chick feather and scale rudiments

To begin to study the role of particular proteins in inductive tissue interactions, we have used density labelling techniques to determine whether any dermal proteins are found between embryonic chick dermis and epidermis at a stage when the dermis plays an important inductive role in epidermal differentiation. Epidermis will form feathers or scales depending on whether it interacts with dorsal or foot dermis, respectively, and the dermis can still influence epidermal differentiation when direct cell contact between the tissues is blocked by a membrane filter during culturing (Peterson & Grainger, 1985). In transfilter experiments, we detect a subset of dermal proteins within the filter between the tissues. Several of these dermal proteins are deposited in a region-specific manner, that is, they are only found associated with filters from either dorsal or foot dermis. We have previously shown that the expression of some of these proteins is specific to particular regions of dermis and is also associated with the inductive potential of the dermis (Peterson & Grainger, 1986). We detect only 17 dermal proteins which are transferred across the filter in these cultures and found in direct association with epidermis; of these 14 are common to both dorsal and foot dermis, and 3 are deposited in a region-specific manner. Our results lead us to hypothesize a significant function for certain dermal proteins in this inductive interaction either as part of the extracellular matrix or in direct association with epidermis.     

The structural periodicity ofE. coli ribosomal proteins

It is established that the sequences of all different proteins fromE. coli ribosome as well as two protein biosynthesis initiation factors, two ribosome-associated DNA-binding proteins, and the elongation factor EF-Tu from the same source possess a periodicity expressed more weakly and different from that found earlier for a number of proteins representatives of 18 superfamilies. The statistical significance of the periodicity observed was checked by comparing the area below the periodicity curve of every protein examined with that of computer generated sequences having the same amino acid composition and length. The results concerning the proteins from small and large ribosomal subunit are compared. The conclusions support and supplement the concept about the presence of a trend in protein molecular evolution from universal (Gly, Ala) to specialized (Phe, Tyr, Trp, Cys) amino acids.     

Junin virus structural proteins.

Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.     

Bovine coronavirus structural proteins.

The tissue culture-adapted strain (Mebus) of bovine coronavirus was grown in the presence of isotopically labeled amino acids, glucosamine, or orthophosphate for the purpose of analyzing the virion structural proteins. Five species of polypeptides were identified when purified virions were solubilized in urea and sodium dodecyl sulfate and resolved by polyacrylamide gel electrophoresis. Four species were glycosylated and had apparent molecular weights of 140,000, 120,000, 100,000, and 26,000. The glycoproteins were susceptible to proteolytic cleavage and enzymatic iodination when intact virions were studied and are thus at least partially external to the virion envelope. The 140,000-molecular-weight glycoprotein is apparently a dimer of 65,000-molecular-weight glycopolypeptides held together by disulfide linkages. Species 5 was phosphorylated and had an apparent molecular weight of 52,000. In the intact virion, it was unaffected by protease and was not enzymatically iodinated. It is therefore apparently an internal protein.     

Fine structural observations on the epidermis

Summary The organization of the wall of epidermal cells in the petiole of species of Apium, Eryngium, Rumex, and Abutilon as well as that of the epidermis of Avena coleoptile has been investigated. The outer and inner tangential walls consist of layers in which the cellulose microfibrils are oriented alternately parallel or transverse to the longitudinal cell axis. This organization resembles that previously described for collenchyma cell walls (Wardrop, 1969; Chafe, 1970). On the radial (anticlinal) walls the orientation of the microfibrils is transverse and these appear continuous with the layers of transverse orientation of the outer and inner tangential walls. Variation in thickness of the outer tangential, and radial, and inner tangential walls appears to result from the variation in thickness of those layers in which the microfibrils have a longitudinal orientation. The extent to which these observations can interpreted in terms of some type of modified multi-net growth is discussed.     

Fine structural observations on the epidermis

Summary In species of Apium, Eryngium and Humulus, the cuticular membrane of the petiole could be resolved into two parts, of which the inner one appeared amorphous and after staining appeared to be penetrated by an electron-dense reticulum, whereas the outer layer showed a lamellate structure consisting of electron-dense and electron-transparent plates, 50–80 Å in thickness. These layers are considered to correspond with the cuticular layer and the cuticle proper, respectively. In species of Abutilon and Rumex the cuticle proper did not exhibit the lamellate structure. In the leaves of Eryngium the outer lamellated structure was present in the cuticle of both young and mature leaves. Both the lamellate and non-lamellate types of the cuticle proper increased in thickness with age of the specimen. The results are discussed in relation to earlier investigations.     

Exploring structural homology of proteins.

A method for systematically comparing the folding of the three-dimensional structures of proteins has been developed. A search function, plotted in terms of three Eulerian angles, represents the number of sequentially equivalenced amino acids. For each orientation one protein structure is rotated about its center of mass with respect to the other and probabilities are calculated which estimate the degree of structural parallelism. The structurally equivalent residues with highest probabilities are then selected for the best common topology. It was observed that, when structures containing about 150 residues were compared, the random background had a mean value of around 14 residues and the standard deviation was approximately nine residues. The method has been shown to be successful in determining the similarity of the NAD binding domains of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, and in comparing the heme binding fold of cytochrome b₅ with the globins.Application of the method to compare hen egg white lysozyme and T4 phage lysozyme led to a single significant peak of 62 residues. The structural homology indicated by this peak showed that the substrate, as bound to hen egg white lysozyme, has a corresponding binding site in the large cleft of the phage lysozyme. The predicted binding site of N-acetyl glucosamine at position C compares well with an N-acetyl glucosamine center observed to bind to crystalline phage lysozyme (B. W. Matthews, personal communication).Some results for the comparison of the two Fe-S cage binding domains of ferredoxin are also presented.     

The assessment of post mortem structural changes in the human epidermis

The structure of epidermis and appearance of keratinocytes is described in intact skin specimens from human corpses stored after death under refrigeration. Two groups of alterations can be identified depending on the epidermal layer. In the spinous layer, the cells are characterized by crescent-shaped nuclei surrounded by a hollow area. The number of such cells increases significantly each day during the first 8 days post mortem (dpm), and their frequencies follow respective regression equations, so as to enable the post mortem time estimation with one day accuracy. In the basal layer, distorted, balloon-shaped cells with pycnotic nuclei appear, which with the lapse of time are forming groups, and eventually the epidermis in those places separates from the dermis. The presence of both described changes seems to indicate whether the skin sample was obtained from the living organism or after the death.     

The tree structural organization of proteins

We offer an objective definition of the domains of a protein, given its C^α coordinates from high-resolution X-ray crystal studies. This is done by an algorithm which groups segments of the polypeptide chain together when there are a relatively large number of contacts between the two segments. The result is an organizational tree showing a hierarchy of segments grouping together, then clusters merging until all parts of the chain are included. In this view the highest level clusters correspond well to more subjective definitions of folding domains and the lowest level, the segments, roughly match the usual assignments of pieces of secondary structure. The intermediate level clusters suggest possible folding mechanisms, which are discussed.     

Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.     

Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.     

The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.