Proteoglycans in cultures of skin fibroblasts in recessive dystrophic epidermolysis bullosa.

The proteoglycans of cultured fibroblasts from the skin of three patients with recessive dystrophic epidermolysis bullosa and three normals were compared after labeling with [35S]sulfate and [3H]leucine. The behavior in gel chromatography of the intact proteoglycans and several properties of their component glycosaminoglycans (size, content of iduronic acid, and content of 4- and 6-sulfate) showed no statistical differences. In addition, the binding of intact proteoglycans and of their constituent proteins and glycosaminoglycans to type I collagen were measured by affinity chromatography. No differences were found that could account for the skin lesions in recessive dystrophic epidermolysis bullosa.

Biochemical analyses of proteoglycans in rabbit corneal scars.

Macromolecules from normal rabbit cornea and cornea containing a 2-mm diameter button of scar tissue were biosynthetically labeled with 35S-sulfate and 3H-glucosamine in vivo and in organ culture. Labeled macromolecules, including proteoglycans (PGs) extracted from the normal cornea, scar tissue, and corneal tissue adjacent to the scar with guanidine hydrochloride were chromatographed on DEAE-Sepharose CL-6B columns and eluted with increasing concentrations of NaCl. The elution pattern of corneal macromolecules synthesized in vitro was remarkably similar to that in vivo. In another experiment, corneas having 2-, 4-, and 8-week-old scars were labeled in organ culture and also extracted. Scars synthesized PGs with lower sulfation than those of adjacent corneal tissue. Although PG synthesis in scar decreased with wound age, the synthesis in adjacent cornea remained the same. In a third experiment, PGs extracted from pools of unlabeled 2- and 4-week-old scars, adjacent corneal tissue, and normal corneas were chromatographed on ion-exchange columns and analyzed chemically. The quantity of PGs in scar and adjacent cornea increased with healing time. The ratios of keratan sulfate PG to dermatan sulfate PG in normal cornea, scar, and adjacent cornea was 2.3, 0.6, and 1.5, respectively. The PGs from adjacent corneal tissue had a higher charge density than those from scar. The predominant adjacent-cornea dermatan sulfate PG had a higher charge density than that in normal cornea. The authors conclude that cornea adjacent to the healing wound synthesized PGs measurably different fro those in scar and normal cornea.

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy.

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.

Secondary structure of a core protein from pig skin proteodermatan sulfate: CD and Fourier transform IR spectroscopic studies in solution.

The secondary structure of a 38 kDa core protein from pig skin proteodermatan sulfate (PDS), was investigated in solution using CD and Fourier transform (FT) ir spectroscopy. Both techniques generally have provided complementary data on the secondary structures of proteins. CD spectral analysis has shown that the core protein contains 60% beta-turn and alpha-helical structures, the rest being "unordered" structure. FT ir data do not permit calculation of quantitative contributions of substructures, at the present time, to the overall secondary structure of the core protein. CD spectrum of the intact PDS is similar to the core protein CD spectrum.

Secondary structure of pig skin proteodermatan sulfate: a perspective from Raman spectroscopic studies in aqueous solution.

Raman spectroscopic studies of pig skin proteodermatan sulfate in H2O are indicative of a well defined secondary structure consisting of alpha-helical, beta-turn, and possibly "random" structures. The above conclusion is surprisingly close to the secondary structure of the "core" protein of pig skin proteodermatan sulfate proposed in the previous paper (V. Renugopalakrishnan et al., Biopolymers 28, 1923-1933, 1989) from FT-IR and CD spectroscopic studies in aqueous solution.

Developmental changes in proteoglycans of rabbit corneal stroma.

Proteoglycans have been extracted from rabbit corneal stromas at developmental stages from fetal to adult. Ion exchange fractionation and gel chromatography show that proteodermatan sulfates decrease in sulfation and relative amount and proteokeratan sulfates increase in sulfation and relative amount during development. There are small increases in size of all of the proteoglycans up to 2 weeks after birth, and the final adult composition is achieved by 8 weeks.

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits.

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.

Sera from patients with poststreptococcal glomerulonephritis contain antibodies to glomerular heparan sulfate proteoglycan.

Antibodies, found in human sera from patients with poststreptococcal glomerulonephritis, against proteoglycans (PG) derived from bovine and human glomeruli were investigated. PG were isolated by 4 M guanidine-HCl extraction of whole glomeruli, followed by DEAE-Sepharose CL-6B ion exchange chromatography. The anionic fractions were further purified by chromatography on Sepharose CL-4B. Biochemical analysis of the two resulting peaks revealed the presence of high molecular weight anionic material containing protein, uronic acid, glucosamine, and galactosamine. Enzymatic and chemical susceptibilities indicated the presence of heparan sulfate PG and a galactosamine-containing PG. Immunologic studies revealed the presence of anti-PG antibodies to both PG peaks of the Sepharose CL-4B column in glomerulonephritis sera. Inhibition studies using an ELISA demonstrated that heparan sulfate was a major antigenic determinant. Cross-reactivity with both mammalian and streptococcal hyaluronate was noted. Inhibition studies also indicated the presence of a second antigenic site containing N-acetylgalactosamine, possibly representing chondroitin or dermatan sulfate PG.

Monoclonal antibodies to proteokeratan sulfate of rabbit corneal stroma.

Hybridomas were developed that secreted monoclonal antibodies against two proteokeratan sulfates (PKS) from rabbit corneal stroma. A total of 28 antibodies were isolated, all of the IgG type with kappa light chains. All were found to react with both PKSs. As judged by immunohistochemical staining, none of them reacted with scleral or conjunctival components, nor with sections of skin, but all reacted with nasal cartilage. When tested by enzyme-linked immunosorbent assays, against components of the proteoglycans, all of the antibodies reacted with keratan sulfate-peptide (isolated from papain digests of PKS or of cartilage proteoglycan), and all but two reacted with oligosaccharide-containing protein cores (prepared by keratanase treatment of PKS). Reactivity with cores was probably due to residual portions of the keratan sulfate chains since the endogenous oligosaccharide-peptides (non-sulfated, non-keratan sulfate oligosaccharides isolated after papain digestion of PKS) were not active. None of the antibodies reacted with protein cores made by removal of carbohydrate by hydrolysis with trifluoromethanesulfonic acid. All except one reacted with fragments of keratan sulfate (made by keratanase digestion). These observations are evidence for structural requirements at three different levels of completeness of the antigen for recognition among the various antibodies. In addition, none of the antibodies reacted immunohistochemically with macular dystrophic corneas, confirming the finding of others that the defect lies in the keratan sulfate portion of the proteoglycans.

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core.

Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues.

Proteoglycans of rabbit cornea: labelling in organ culture and in vivo.

Rabbit corneas maintained with radioactively-labelled precursors in organ culture for up to 42 hr produced labelled proteoglycans of the same kind as those that exist normally or that are produced by labelling in vivo. Whole corneas, including a narrow strip of sclera, were kept in culture in the presence of [3H]-glucosamine and [35S]-sulfate. The rate of incorporation of sulfate into extractable proteoglycans was linear over the time investigated, as was the rate of incorporation of glucosamine after a short lag. Three labelled proteoglycans were isolated and found to behave in ion-exchange chromatography and gel chromatography in the same way as they did in previous studies by chemical analysis. Their labelled glycosaminoglycans were primarily dermatan sulfate and keratan sulfate, with traces of hyaluronic acid and heparan sulfate. When labelled precursors were injected directly into the anterior chamber of rabbit eyes, the resulting labelled proteoglycans were similar to those obtained in organ culture. Both in vivo and during organ culture, the specific activity of hexosamine in the keratan sulfate proteoglycans was about one-half that in dermatan sulfate, probably because of different synthetic rates or different specific activities of immediate precursors.

Proteoglycans of rabbit corneal stroma. Isolation and partial characterization.

Proteoglycans extracted from rabbit corneal stroma can be separated by ion exchange and gel chromatography into two proteokeratan sulfates (PKS-I, PKS-II) and two proteodermatan sulfates (PDS-I, PDS-II). PKS-I (21% of the total glycosaminoglycans) contains 48% protein, and PKS-II (43% of the total) has 57% protein. In both, the only hexosamine is glucosamine, 7% of which is found in oligosaccharides. There is much more sialic acid in the oligosaccharides of PKS-II than of PKS-I, and the amino acid compositions of the two proteoglycans differ significantly. The keratan sulfates isolated from them by papain digestion are different in size, but both are digested by endo-beta-galactosidase. PDS-I (30% of the total glycosaminoglycans) has 32% protein, and 35% of its uronic acid is iduronic acid. PDS-II (6% of the total) has 42% of its uronic acid as iduronic. In general, the four rabbit proteoglycans resembled those of human and bovine corneal stroma in being smaller and more protein-rich than those of cartilage.

Proteodermatan sulfate isolated from pig skin.

The major proteoglycan of pig skin, a proteodermatan sulfate, has been isolated under mild conditions in the presence of protease inhibitors. After purification by ion exchange and gel chromatography, it has a Mr of 70 x 10(3) and it does not form aggregates with hyaluronic acid. It contains about 60% protein, one chain of dermatan sulfate, and several oligosaccharide chains. The dermatan sulfate is high in iduronic acid (85% of the uronic acid) and there are no chains of chondroitin sulfate present. The oligosaccharides contain glucosamine, sialic acid, and neutral sugars.

Plasmid-related transmissibility and multiple drug resistance in Streptococcus faecalis subsp. zymogenes strain DS16.

Streptococcus faecalis subsp. zymogenes strain DS16 was found to harbor two plasmids, designated pAD1 (35 megadaltons) and pAD2 (15 megadaltons). pAD1 is transmissible and determines a hemolysin-bacteriocin, whereas pAD2 is non-conjugative and determines resistance to erythromycin, streptomycin, and kanamycin. pAD2 could be mobilized by pAD1, but usually involved formation of a pAD1-pAD2 cointegrate.