Basement membrane collagen synthesis by rabbit corneal endothelial cells in culture. Evidence for an alpha chain derived from a larger biosynthetic precursor.

Corneal endothelial cells in culture synthesize basement membrane collagen and secrete it into the medium. This collagen sediments faster than interstitial collagen by velocity sedimentation and is disulfide-bonded. After reduction, two biochemically distinct chains can be determined by cyanogen bromide peptide mapping. These chains migrate close to each other and immediately below beta 12(I) components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with pepsin gives rise to a major band which still retains interchain disulfide bonds and which will not convert to components with the mobility of interstitial alpha chain by reduction. However, an alpha chain and three minor collagenase-sensitive and pepsin-resistant peptides are generated if the molecule is reduced and alkylated under nondenaturing conditions prior to pepsin treatment. When collagen which accumulates in the media over a long period of time is compared to the newly synthesized molecules, there is an apparent differential resistance to limited pepsin treatment. However, the products which are generated in both cases share electrophoretic identity.

( 1 Correspondence may be addressed to the Department of Ophthalmology at the university to P.O. Box 60132, Terminal Annex, Los Angeles, CA 90060. digests of anterior lens capsule, glomerular, and placental membranes (4)(5)(6)(7). Sato and Spiro (8) found in glomerular basement membrane a heterogeneous mixture of collagen components ranging from M, = 25,000 to 205,000 which they extracted in the absence of proteases. Bornstein and co-workers (9, 10) characterized type IV procollagen from human amniotic ceUs in culture which contains two biochemically distinct chains which migrate between p components and pro a1 (I) chain. Procollagen molecules of similar size were isolated from anterior lens capsule (11)(12)(13). Timpl et al. (14) isolated basement membrane collagen-like materials considerably larger than a chains from EHS tumors not subjected to protease treatment.
Although there is no universally acceptable molecular model for basement membrane collagen, the variously described materials share common characteristics distinct from interstitial collagens. They have elevated but variable levels of 3-hydroxyproline, elevated ratios of 4-hydroxyproline to proline and hydroxylysine to lysine, and low alanine and arginine contents. In addition, hydroxylysine is highly glycosylated with galactose or glucosylgalactose.
The in vitro biosynthesis of basement membrane collagen has been demonstrated in several systems (9, 10, [15][16][17]. The corneal endothelium is a single layer of cells covering Descemet's membrane on the most posterior surface of the cornea of the eye. In this paper, we report the synthesis of basement membrane collagen by corneal endothelial cells in culture and present data indicating that these cells produce two high molecular weight collagenous polypeptides which can in turn generate an a chain like product.

Culture of Rabbit Corneal Endothelial
Cells-Isolation and establishment of corneal endothelial cells in culture were previously described (18). Primary cultures, which were maintained in DME medium' (Gibco) supplemented with 10% fetal calf serum (Gibco) and 50 p g / d of gentamicin, were used throughout the experiment. All cultures were incubated at 37 "C under a humidified atmosphere of 7.5% COS in air. Cells grown to confluence in 100-mm plastic culture dishes (Falcon) were labeled for 24 h with 10 ml of DME medium containing 500 pCi of [5-3H] cystine (371 Ci/mM) (Amersham), 2% fetal calf serum, 64 pg/ml of 2amino propionitrile, and 100 p g / d of ascorbate. Medium was collected and the following protease inhibitors were added: 1 rmf phenylmethylsulfonyl fluoride, 10 m~ N-ethylmaleimide, 4 mM ethylenedinitrilotetraacetic acid, and 0.1 mM a,d-dipyridyl. Ammonium sulfate was added to the medium to 45% of saturation after centrifugation The abbreviations used are: DME medium, Dulbecco-Vogt-moditied Eagle's medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CNBr, cyanogen bromide; NCP, noncollagenous protein.

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for removal of cell debris. The precipitate was collected by centrifugation at 10,000 X g for 15 min at 4 "C and dissolved in 2.0 M urea and Buffer I (0.05 M Tris-HCI, 0.15 M NaCI, and 0.1% Triton X-100, pH 7.4) followed by dialysis into the same buffer.
Velocity Ultracentrifugation-The sample was layered on a 5 to 20% sucrose gradient containing 2.0 M urea and Buffer I placed over a 4070 sucrose cushion and sedimented in a Beckrnan SW 40 rotor at 40,000 rpm for 48 h at 4 "C. The gradient was then fractionated. Fractions containing collagenous proteins were pooled and dialyzed against 6.0 M urea in Buffer I at 4 "C. The samples were denatured by heating to 55 "C for 30 min and sedimented on a 5 to 20% sucrose gradient containing 6.0 M urea and Buffer I in a Beckrnan SW 40 rotor at 40,000 rpm for 48 h at 20 "C. SDS-Polyacrylamide Slab Gel Electrophoresis-Polypeptides were electrophoresed under the conditions described by Laemmli (19) and fluorograms were developed by Kodak RPX-Omat processor (20,21).
Peptide Mapping by CNBr Digestion-CNBr digestion was carried out as described previously (22) with some modifications. Protein bands localized in slab gels by fluorography were cut out of the dried gel. These were rehydrated in 70% formic acid and digested with 1.0 M CNBr for 4 h at 30 "C. At the end of the digestion, the gel pieces were dried down in a water bath under bubbling Nz gas and washed with distilled water three times. They were then placed into the slot of a 3% stacking gel and electrophoresed on a 10% running gel.
Analysis for Hydroxyproline-The hydroxyproline content of the sample was determined after hydrolysis in 6 N HCl under vacuum at 110 "C for 24 h. The hydrolysate was dried and then dissolved in 0.01 N HCl. It was then applied to a short column of a JEOL amino acid analyzer (JLCdAH). Elution was carried out with 0.2 M sodium citrate buffer, pH 2.81, at 35 OC. Each fraction was counted in a Beckman LS 3133 T counter.
Reduction and Alkylation under Nondenaturing Conditions-Partially purified sample from sucrose velocity sedimentation was dialyzed against 0.1 M Tris-HCI, 0.15 M NaCI, and 2.0 M urea, pH 7.5, at 4 "C and degassed. Dithiothreitol (final concentration, 20 mM) was added and the reaction mixture was stored for 4 h at 4 "C. Iodoacetamide (final concentration, 80 RIM) was then added followed by incubation for 24 h at 4 "C.
Enzyme Digestions-Samples were incubated with 100 pg/ml of pepsin for 24 h at 4 "C or 24 h at 15 "C or 22 "C. Pepsin activity was stopped by raising the pH to 8.0 by addition of solid Tris and boiled for 1 min in the presence of 0.2% SDS. Protease-free bacterial collagenase digestion was performed as described by Peterkofsky and Diegelman (23). The reaction was stopped by addition of SDS to a final concentration of 0.2% followed by boiling.
Preparation of Marker Collagen-Fibroblasts from embryonic rabbit skin were cultured by conventional techniques (24). Secondary cultures were then labeled and the collagen purified (24).

RESULTS
Isolation a n d Purification of Basement Membrane Collagen-Basement membrane collagen synthesized by the corneal endothelial cells was recovered from the medium. Collagenous proteins accounted for approximately 12 to 15% of the total nondialyzable ['Hlproline-labeled protein. Initial isolation was performed by ultracentrifugation under native conditions. A typical sedimentation profile is shown in Fig. lA. Brief preincubation of the ammonium sulfate precipitate prior to sedimentation with bacterial collagenase partially removed peak I and almost completely peak 11. Peak I sedimented faster than a type I collagen marker (Fig. IA). Limited pepsin treatment of peak I material rendered a disulfide-linked collagen reducible to one a1 size band (data not shown). These results suggest that peak I may contain a precursor form of type I11 collagen (Fig. 1B, arrow). Peak I1 is rich in two closely spaced collagenous bands migrating immediately below p components and fibronectin. These were designated as CE-1 and CE-2. Similar migrating bands have been reported in human amniotic fluid cell culture systems (9, 10). One of the components of peak I11 was identified as fibronectin by immunoprecipitation technique (antibody against fibronectin was provided by Dr. Erkki Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA).

Membrane Collagen
The materials from peak I1 were pooled and further purified by ultracentrifugation under denaturing conditions. The sedimentation profile of the denatured CE collagen was compared with type I11 collagen. Denatured type I11 collagen whose molecular weight is about 300,000 sedimented to fraction 25, while denatured CE collagen sedimented much faster (Fig. 2). Type I11 collagen contains intrahelical disulfide bridges near the carboxyl-terminal end (25, 26). The unusually fast sedimentation of denatured CE collagen suggests that in addition to its larger molecular sue, it may represent a more compact structure than type I11 collagen. When fractions 36 and 37 were analyzed on SDS-PAGE under reducing condition, the characteristic CE-1 and CE-2 bands were the only peptides present, suggesting that this is a good method for purification.
When cystine-labeled medium fraction was analyzed on SDS-PAGE after ultracentrifugation, noncollagenous, pepsinsensitive, proline-poor, and cystine-rich protein co-sedimented with CE collagen. This molecule migrated to the position of al(1) prior to reduction (not shown). However, after reduction, it migrated slower (Fig. IC, NCP). Characterization

of the Basement Membrane Collagen-
When the materials from peak I1 in Fig. l A were incubated with pepsin at 4 "C or 15 "C for 24 h, the electrophoretic mobility of the major collagenous protein was unaltered (Fig.  3A, inset, lanes 3 and 4). Reduction often generated a heavy band which migrated just below standard CE-1 and a very faint band just below CE-2 (Fig. 3A). Although this treatment also generated a low molecular weight fragment (Fig. 3A,  inset, arrow), there was no conversion of the basement membrane collagen to components with the mobility of interstitial a chains. When the materials from peak I1 were reduced and alkylated under nondenaturing conditions, the reduced and alkylated molecules also failed to show a change in the electrophoretic mobility (Fig. 3B). However, pepsin digestion of the reduced and alkylated molecules generated an a chain and three low molecular weight collagenase-sensitive fragments (Fig. 3B). In order to study whether the a chain has pepsinsensitive sites and is able to generate these low molecular collagenous fragments, prolonged digestion at a high enzyme to substrate ratio and at elevated temperatures (15 "C and 22 "C) was used. An essentially identical profile of collagenous peptides was recovered under all experimental conditions tested (Fig. 4).
Since one of the characteristics of basement membrane collagen is that of a elevated ratio of hydroxyproline to proline, the extent of hydroxylation of [3H]proline was determined.   Samples from the various steps were analyzed (Table I). Peak I1 material in Fig. lA showed 36% hydroxylation, whereas the same sample followed by pepsin digestion showed 45%. When peak I1 was analyzed after reduction and alkylation under nondenaturing conditions, approximately 42% of the nondi-alyzable 3H activity was recovered as.hydroxy['H]proline. The sample recovered by the ultracentrifugation under denaturing conditions contained about 43% of its imino acid as hydr~xy[~H]proline.
In order to study whether CE-2 might be a proteolytic degradation product of CE-1, both polypeptides were separately digested with cyanogen bromide. The resulting peptides were resolved by electrophoresis. Since major CNBr peptides from CE-1 (Fig. 5, arrows) are not present in CE-2 peptides, this excludes the possibility that CE-1 and CE-2 have common O r i g i n s .
Since pepsin treatment of the newly synthesized CE collagen fails to generate components with the mobility of interstitial a chains, it seemed of interest to examine the characteristics of CE collagen accumulated in the media over a long the "C-labeled medium was recovered and the cells incubated with [3H]proline for another 24 h. The accumulated CE collagen was compared with the newly synthesized molecules after pepsin digestion. The major products observed on SDS-PAGE were essentially identical with those generated by the newly synthesized molecules in terms of electrophoretic migration, suggesting that CE collagen appeared not to be significantly altered in size (Fig. 6, lunes 2 and 3). However, it should be noted that the ratio of CE-lp to CE-2p (the respective degradation products of CE-1 and CE-2) in the newly synthesized molecules is much greater than that of the accumulated collagen. This observation demonstrates differential susceptibility of CE-1 and CE-2 to proteolytic modification.

DISCUSSION
For more than a decade, a genetically distinct collagen type with chain composition [a1(IV)]3 has been proposed as the major component of basement membrane. Nevertheless, there is no generally acceptable molecular model, and it is not known whether basement membrane collagen is a family of different peptides from one or various distinct genetic types.
Rabbit corneal endothelial cells in culture synthesize and secrete mostly basement membrane collagen (CE) and a precursor form of type 111 collagen as a minor component. The electrophoretic behavior and the degree of susceptibility to limited pepsin treatment of CE collagen are similar to AF, procollagen synthesized by human amniotic fluid cells (9, 10) and endothelial cells (27,28). Like these collagens, CE collagen migrated as distinct chains just below collagen p components upon reduction. Pepsin treatment yields one or two chains which migrate just below the position of CE collagen upon reduction (Fig. 3). Although CE collagen appears as a doublet on SDS-PAGE after reduction, the ratio of the two bands, CE-I/CE-2, is much greater than 2 but fluctuates with the conditions of purification.
The triple-stranded disulfide-linked CE collagen can be pwified by velocity ultracentrifugation, and comparison of the sedimentation profiles under denaturing conditions with type 111 collagen demonstrates its faster sedimenting behavior. This may be attributed to two factors: 1) the larger molecule size of CE collagen compared to collagen, and/or 2) a more globular (or compact) conformation of CE collagen due to disulfide bonds at both ends (probably involving noncollagenous extensions).
It is of interest to note that a noncollagenous, pepsin-sensitive, proline-poor, and cystine-rich protein (Fig. IC, NCP), whose molecular weight is approximately 100,000 as determined by electrophoretic mobility, co-sedimented with CE collagen. This NCP is heavily labeled by cystine and appears to contain intramolecular disullide bonds which upon cleavage generate a molecule of larger hydrodynamic volume. This observation suggests two possibilities. 1) This NCP may form aggregates as judged by its sedimentation behavior and these aggregates sedimented coincidentally with CE collagen; and 2) this NCP may be associated with this collagen and explain the different recovery and inconsistent appearance of the two closely spaced chains of CE collagen when an ammonium sulfate precipitate was examined on SDS-PAGE.
The extent of hydroxylation of proline residues in CE collagen is lesser than other basement membrane collagens. This difference may suggest three possibilities. 1) CE collagen has a low degree of hydroxylation; 2) underhydroxylation may be associated with the tissue culture conditions; and/or 3) other covalently linked peptides may protect the molecule from enzymatic attack, thus, allowing CE collagen to retain noncollagenous extensions. Therefore, the hydroxyproline content becomes diluted by these noncollagenous sequences. On the other hand, the fact that highly purified CE collagen by pepsin treatment or from the reduced and alkylated material followed by pepsin treatment contains low hydroxyproline values seems to rule out the latter possibility. The substantial differences in CNBr peptide profiles rule out the possibility that CE-2 might originate from a common precursor.
Dehm and Kefalides (3) generated an a size chain from bovine lens capsule by a procedure which includes two-step proteolysis by pepsin. The collagen is indistinguishable from al(I) chain on SDS-PAGE. Miller and other groups (4-7) reported two biochemically distinct chains, C chain (a size) and D chain (80,000) differing in ionic charges and CNBr cleavage patterns. On the other hand, larger peptides which migrate just below p components on SDS-PAGE after reduction were recovered from the several tissue culture systems (9,10,27). The present study provides evidence that an a like chain is derived from the high molecular weight chains which migrate just below , 8 components. Limited pepsin digestion of the intact CE collagen does not convert the molecule to components with the mobility of interstitial a chains, and it 7120 Corneal Basement Membrane Collagen seems that reduction and alkylation prior to pepsin digestion are necessary to generate such a product. It has been reported that an a size chain can be isolated from lens capsule subjected to partial limited proteolysis before and after reduction under nondenaturing conditions (3). Therefore, it is clear that partial reduction is necessary for the enzyme to cleave the basement membrane collagen into a size chains.
Since newly synthesized CE collagen does not respond to pepsin in the same manner as interstitial procollagens, the reasons were further investigated. When newly synthesized CE collagen was labeled with C3H]proline for 24 h, the ratio of the resulting peptides after pepsin treatment, CE-lp/CE-2p, varies. In all cases, however, the intensity of CE-lp is much greater than that of CE-2p, suggesting not only that CE-1 and CE-2 are distinct molecules but that the newly synthesized CE-2 is much more sensitive to limited proteolysis. On the other hand, when CE collagen was labeled to accumulate for 13 days in culture, the CE-lp and CE-2p were recovered in similar amounts (Fig. 6, lane 3). The fact that the stoichiometry of the CE-1 and CE-2 chains is greater than 2:l and that they exhibit a differential susceptibility to limited proteolysis is further evidence that CE-1 and CE-2 are distinct species. One can speculate that the a size chains obtained from pepsin digestion of the reduced and alkylated peak I1 materials (Fig.  3B) may be derived from CE-1 (in contrast to the small molecules which probably originate from the pepsin unstable CE-2). Although further evidence is needed to support this speculation, single basement membrane collagen species containing two chains cannot be ruled out.
It has recently been reported that type I11 collagen was the major collagenous component in bovine corneal endothelial cell culture (29). Our discrepancy with these findings may be due to the culture age (primary uersus late passages), animal species (rabbit uersus bovine), or the culture conditions (absence versus presence of growth factors). It should be noted that type I11 collagen in the bovine system was determined by only DEAE-and carboxymethyl (CM)-cellulose column chromatography, while electrophoretic mobility, sedimentation behavior, and CNBr peptide patterns were used in the present study. Rabbit corneal endothelium in organ culture incubated with radioactive precursor synthesizes only basement membrane collagen (16). However, type I11 procollagen is synthesized as a minor collagenous component in rabbit corneal endothelial cells in culture. Immunofluorescent studies with antitype IV antibody ( g f t of Dr. George Martin, National Institutes of Health, Bethesda, MD) and anti-type 111 antibody show that rabbit Descemet's membrane only stains with anti-type IV antibody? Presently, it is not clear whether type I11 collagen is a component of Descemet's membrane.
These results may provide a link between the two electrophoretically distinct species of basement membrane collagens described in the literature, namely CE-1 and CE-2 collagen, AF1 procollagens, and C and D chain.