Post-translational Modifications of the Core-specific Lectin RELATIONSHIP TO ASSEMBLY, LIGAND BINDING, AND SECRETION*

The rat core-specific lectin (CSL) or mannan-binding protein is synthesized and secreted by rat hepatocytes and H-4-11-E hepatoma cells. Prior to secretion proline and lysine residues with collagen-like sequences undergo hydroxylation and subsequent glycosylation of hydroxylysine to produce glucosylgalactosylhy- droxylysine. Hydroxylation and subsequent glycosylation are inhibited by a,d-dipyridyl (Colley, K. J., and Baenziger, U. U. (1987) J. Biol. Chern. 262, 10290- 10295). We have used a,d-dipyridyl to investigate the role of hydroxylation and glycosylation on interchain disulfide bond formation, assembly of subunits into high molecular weight complexes, attainment of car- bohydrate and lipid binding ability, and secretion. Formation of disulfide-bonded dimers and trimers in the endoplasmic reticulum, assembly into high molecular weight complexes in the Golgi, and attainment of car- bohydrate binding activity occur in either the presence or absence of these post-translational modifications. The mature fully processed form of the CSL binds hydrophobic matrices and is secreted at a slow, but linear, rate. Inhibition of proline and lysine hydrox- ylation and hydroxylysine glycosylation prevents CSL secretion and attainment of binding activity for hydro- phobic matrices. Secretion of the lectin, although slow, appears

its ability to recognize features of the "core" region of asparagine-linked oligosaccharides and to bind to yeast mannans, respectively (1). The CSL is probably identical to the rat mannan-binding protein (MBP-C) recently characterized by Drickamer et al.
(2). We previously observed that the CSL has a half-time for secretion of >4 h and that the lectin is retained within the Golgi prior to secretion from the cell (3). The CSL undergoes two distinct post-translational modifications which are detected during analysis by SDS-PAGE as successive increases of 1,000 in M , resulting in an increase in the total M, from 24,000 to 26,000. The subunits are also assembled into a high molecular weight multimeric complex prior to secretion (4). The post-translational modifications and/or assembly of subunits into the high molecular weight form are required for attainment of carbohydrate binding activity by the CSL (4). Although these events all appear to take place within the Golgi, they are completed 60-80 min after the initiation of synthesis, and their completion does not account for the prolonged retention of the lectin within the cell prior to secretion (4).
(2) have reported that the rat MBP-C contains collagen-like sequences and have demonstrated the presence of hydroxyproline in lectin isolated from rat liver. In the accompanying paper (5) we have demonstrated that CSL synthesized by cultured rat hepatoma cells contains both hydroxyproline and hydroxylysine. The majority of the hydroxylysine residues in the lectin are glycosylated to form glucosylgalactosylhydroxylysine. a,a'-Dipyridyl, an inhibitor of both prolyl and lysyl hydroxylases, prevents the posttranslational increase in M , of the CSL, indicating that hydroxylation and hydroxylysine glycosylation account for the post-translational increases in M, we have previously observed (3, 4).
The complement component Clq (6,7), pulmonary surfactant apoproteins (8)(9)(10)(11), and the 18 S asymmetrical form of acetylcholinesterase (12)(13)(14)(15) also contain collagen-like domains with hydroxylated amino acids. In the case of Clq, glycosylation is also known to occur (6). A feature characteristic of these proteins and collagens is the assembly of smaller subunits into high molecular weight complexes. Collagen undergoes hydroxylation of proline to 4-hydroxyproline, which stabilizes, and is required for, triple helix formation (16)(17)(18). Lysine hydroxylation is required for the stabilization of collagen intermolecular cross-links; however, the role of hydroxylysine glycosylation is less well defined (16)(17)(18). The CSL forms trimeric units linked together by disulfide bonds and later forms higher molecular weight complexes (2, 4), which may involve triple helix formation. The post-translational modifications of the CSL, like those of collagen, may play a role in subunit assembly into high molecular weight complexes. In this study we have used the hydroxylase inhibitor, a,a'-dipyridyl (19), to investigate the impact of proline hydroxylation, lysine hydroxylation, and hydroxylysine gly-cosylation on the disulfide bond formation, complex assembly, attainment of carbohydrate binding activity, attainment of lipid binding activity, and secretion of the CSL from H-4-11-E cells.

Materials
Staphylococcus aureus was kindly provided by Drs. Susan Cullen and Benjamin Schwartz of Washington University School of Medicine, St. Louis, MO. Mixed alkyltrimethylammonium bromide (predominantly C,,H,N(CH,),Br), sodium deoxycholate and a,a'-dipyridyl were obtained from Sigma. Ultrapure electrophoresis-grade agarose was supplied by Bethesda Research Laboratories. GelBond PAG film gel support medium was obtained from FMC Corp. Shaltiel hydrophobic chromatography kit I, agarose-c, series was purchased from Miles-Yeda.
n,a'-Dipyridyl Inhibition of Prolyl and Lysyl Hydroxylases-To inhibit the hydroxylation of proline and lysine residues during metabolic labeling of H-4-11-E cells, a,a'-dipyridyl, a t a concentration of 3.5 mM, was included 1 h prior to and during labeling and chase periods. The concentration of a,a'-dipyridyl used (3.5 mM) was found to effectively inhibit hydroxylation of the CSL (>go%) and resulted in a less than 10% decrease in protein synthesis in the H-4-11-E cells.
Polyacrylamide Gel Electrophoresis-SDS-PAGE was performed according to the method of Laemmli (20) using 10% polyacrylamide gels and radiolabeled products visualized by fluorography (21).
Sucrose Density Gradient Centrifugation-The native molecular weight of the lectin, synthesized by H-4-11-E hepatoma cells in the presence or absence of 3.5 mM a,a'-dipyridyl, was determined by sucrose density gradient centrifugation. Cells were disrupted by repeated freezing and thawing in the presence of 20 mM Tris-HCI, pH 7.8, 200 mM NaCl, 2 mM EDTA to release the CSL. Approximately 1 ml of supernatant was layered on the sucrose gradient which consisted of 1.7 ml each of 5, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0% sucrose in 20 mM Tris-HCI, pH 7.8, 200 mM NaCl, 2 mM EDTA. Centrifugation was carried out in an SW 41Ti swinging bucket rotor (Beckman) for 18 h a t 4 "C and 200,000 X g. Molecular weight markers were separated on parallel gradients, and their positions were determined spectrophotometrically by absorbance a t 280 nm. Gradient fractions were collected using a Buchler Auto Densi-Flow I1 and an LKB Varioperpex pump. CSL was immunoprecipitated from individual gradient fractions, as described above, and immunoprecipitates were analyzed by SDS-PAGE and fluorography.
Charge-shift Electrophoresis-Charge-shift electrophoresis was used essentially as described by Helenius and Simons (22) to determine whether the CSL is capable of binding hydrophobic agents such as detergents. The CSL and galactose binding protein (GalBP) were isolated from rat serum and liver as previously described (1,23). Protein samples were lyophilized and redissolved in 30 pl of running buffer (0.05 M glycine-NaOH, pH 9.0, 0.1 M NaCI, 0.5% Triton X-100) or in running buffer containing either 0.05% cetyltrimethylammonium bromide (CTAB) or 0.25% sodium deoxycholate. Samples were incubated for 2 h a t 25 "C in the appropriate running buffers. Immediately prior to electrophoresis, 2 pl of 0.05% bromphenol blue, 50% glycerol was added to the protein samples. Running buffer containing the appropriate detergent was used in preparing 1% agarose gels (14 X 12.5 mm) cast on GelBond PAG film gel support medium (FMC Corp.). Samples were loaded in 5 X 7-mm sample wells, and electrophoresis was carried out with a Hoeffer Scientific Instruments MAX Submarine agarose gel unit for 16 h a t 40 V in a 4 "C environment. Instead of submerging the agarose gel, wicks of Whatman No. 3 " chromatography paper were folded over the ends of the gel and placed in contact with running buffer in the anode and cathode chambers. If the proteins bind the detergent the proteindetergent complex migrates in the electric field according to the charge of the detergent. Proteins binding CTAB, a negatively charged detergent, migrate toward the anode, whereas proteins binding deoxycholate, a positively charged detergent, migrate toward the cathode. To detect proteins, the agarose gels were stained with 0.02% Coomassie Blue, 25% isopropyl alcohol, 10% acetic acid, destained with 10% acetic acid, and air dried on the PAG film gel support.
Hydrophobic Chromatography-The hydrophobic character of the CSL synthesized by H-4-11-E cells in the presence or absence of the hydroxylase inhibitor @,a'-dipyridyl was ascertained by hydrophobic chromatography on agarose-clo, according to the method of Shaltiel (24). The hepatoma cells were labeled with 50 pCi/ml ["Hllysine in the presence or absence of 3.5 mM a&-dipyridyl for 24 h. In the presence of a,a'-dipyridyl, unmodified CSL was not secreted and was released from cells by repeated freezing and thawing of cells in 20 mM Tris-HCI, pH 7.8, 2 mM EDTA, 200 mM NaCl. Normally processed mature lectin was isolated from medium following secretion. Both the unmodified lectin and the modified lectin were brought to a final concentration of 25 mM CaC12 and applied to either agarose-cl0 or agarose-co columns equilibrated with 20 mM Tris-HC1, pH 7.8, 25 mM CaCI2, 200 mM NaCI. After washing the columns in the equilibration buffer (4 X 1 ml), bound lectin was eluted with 20 mM Tris-HC1, pH 7.8, 25 mM CaCl,, 200 mM NaCI, 2% Triton X-100 (4 X 1 ml). A final elution with 20 mM Tris-HCI, pH 7.8, 2 mM EDTA, 200 mM NaCl, 2% Triton X-100 (4 X 1 ml) was also included to ensure complete removal of the lectin from the columns. CSL was immunoprecipitated from both unbound and bound fractions, and the immunoprecipitates were analyzed by SDS-PAGE and fluorography.

Hydroxylation of the CSL Is Not Required for Disulfide
Bond Formation-The mature form of the rat CSL migrates on SDS-polyacrylamide gels as dimeric and trimeric species with interchain disulfide bonds (2). The formation of interchain disulfide bonds was examined by SDS-PAGE of immunoprecipitated CSL without prior reduction of disulfide bonds with P-mercaptoethanol ( Fig. 1). We had previously determined that reduction of dimeric and trimeric species yielded the identical monomeric subunit form in each case (see Fig. 2 of Ref. 5). Following 10 min of labeling with ['"S] methionine and 0,10, or 40 min of chase, there is a progressive increase in the amount of CSL migrating as dimer and trimer ( Fig. 1). Trimer is detected after 10 min of chase and is increased in amount after 40 min of chase. The dimeric form is detected even in the absence of any chase period and is increased in quantity a t 40 min as well. The recovery of monomeric forms of the CSL is low at early times. Because similar pulse-chase studies performed in the presence of reducing agents do not indicate poor recoveries at early times, this suggests that the CSL subunits may be associated with another protein(s) prior to formation of subunit interchain disulfide bonds and are either not readily identified following SDS-PAGE or are not efficiently immunoprecipitated. The presence of trimeric as well as dimeric forms of CSL after only 10 min of labeling suggests that interchain disulfide bond formation commences within 10 min after synthesis and is rapidly completed.
The effect of proline and lysine hydroxylation on interchain disulfide bond formation was examined in an analogous fashion ( Fig. 1). Lysine and proline hydroxylation was prevented by exposing H-4-II-E cells to 3.5 mM a,a'-dipyridyl and labeling with [%]methionine for 10 min followed by 0, 10, or 40 min of chase with unlabeled methionine. Trimeric species were detected after 10 and 40 min of chase and dimeric forms even in the absence of any chase period. As was seen in the absence of a,d-dipyridyl, the recovery of monomeric forms of the CSL appears to be low in the absence of reduction. Notably, the trimeric and dimeric forms of the CSL synthesized in the presence of a,a'-dipyridyl have a greater mobility on SDS-PAGE than the same species synthesized in the absence of this hydroxylase inhibitor. This is consistent with the post-translational increase of 2000 in the subunit M , observed upon SDS-PAGE in the presence of reducing agents. Hydroxylation of proline and/or lysine is, therefore, not essential for the formation of disulfide-bonded dimeric and trimeric forms of the CSL.
The formation of interchain disulfide bonds within the first 10 min after initiation of synthesis is in agreement with recent data localizing disulfide isomerase, the enzyme believed to be responsible for disulfide bond formation, in the rough endoplasmic reticulum (25,26). Inhibition of collagen hydroxylation by a&-dipyridyl or anaerobic conditions does not inhibit disulfide bonding between the extension peptides at the carboxyl terminus of interstitial procollagen subunits; however, it does prevent the formation of the triple helix which requires the presence of hydroxyproline (16)(17)(18). Similarly, the hydroxylation of proline and/or lysine in the CSL is not essential for the formation of interchain disulfide bonds. In addition, the rapidity with which interchain disulfide bonds are formed to produce dimeric and trimeric forms of the lectin indicate that disulfide bond formation occurs before the first posttranslational modification detected as an increase of 1000 in the M , of the lectin.
Hydroxylation of the CSL Is Not Required for Complex Assernbly-Prior to secretion from the cell, the CSL is assembled into large complexes of 150,000-260,000 Da when examined by sedimentation on nondenaturing sucrose gradients (4). In H-4-II-E cells, assembly into high molecular weight complexes occurs in the Golgi between 20 and 80 min after initiation of synthesis (4). Only high molecular weight forms of the CSL are bound by mannan-Sepharose, i.e. display carbohydrate binding activity (4). The high molecular weight complexes most likely are formed from disulfide-bonded dimeric or trimeric forms of the CSL, or combinations of both.
To determine if proline and lysine hydroxylation and hydroxylysine glycosylation are required for assembly into high molecular weight complexes, complex assembly was assessed by sucrose density gradient centrifugation of [:%]methioninelabeled CSL synthesized in the presence or absence of 3.5 mM a,a'-dipyridyl (Fig. 2).CSL was immunoprecipitated from individual gradient fractions, and the immunoprecipitates were analyzed by SDS-PAGE and fluorography. The results shown in Fig. 2 demonstrate that CSL synthesized in either the presence or absence of a,a'-dipyridyl migrates as a complex of 150,000 Da in the region of the marker bovine IgG on nondenaturing sucrose density gradients. It is apparent that the a,d-dipyridyl treatment has been effective in preventing hydroxylation and glycosylation since the CSL subunits obtained from treated cells have an M , of 24,000 whereas those obtained from control cells form a diffuse band ranging from M , 24,000 to M, 26,000. Although the amount of CSL obtained from the a,a'-dipyridyl-treated cells is lower than that obtained from the controls in the experiment shown in Fig. 2, this has not consistently been the case (see Fig. 3). In any case, assembly of the CSL into 150,000-260,000-Da native complexes does not require hydroxylation of proline and lysine residues or glycosylation of hydroxylysine.
Hydroxylation of the CSL Is Not Required for Carbohydrate Binding Ability-The CSL or MBP displays calcium-dependent binding of yeast mannan, and mannose and N-acetylglucosamine residues found in the "core" region of asparaginelinked oligosaccharides (1). Binding to mannan immobilized on agarose has been used to purify the lectin by affinity chromatography and to assess carbohydrate-specific binding activity (1,4). Calcium-dependent carbohydrate binding activity is acquired by the lectin synthesized by H-4-II-E cells between 20 and 80 min after initiation of synthesis; at the Eluate fractions from the mannan-Sepharose columns were pooled, concentrated, and applied to nondenaturing 5-20% sucrose density gradients as described under "Experimental Procedures." Samples and molecular weight standards were centrifuged on parallel gradients in an SW 41 Ti swinging bucket rotor for 18 h a t 100,000 X g a t 4 "C. CSL was immunoprecipitated from gradient fractions (1-14), and immunoprecipitates were analyzed by SDS-PAGE and fluorography. Molecular weight standards were detected spectrophotometrically (Amnm). Molecular mass markers: fraction 3, chymotrypsinogen (25.7 kDa); fraction 6, bovine IgG (150 kDa); fraction 12, catalase (232 kDa).
same time the lectin undergoes assembly into high molecular weight complexes and the second stage of post-translational modification. The time course observed for post-translational modification, assembly into high molecular weight complexes, and acquisition of carbohydrate binding activity led us to hypothesize that assembly into high molecular weight complexes and/or the second stage of post-translational modification are required for attainment of carbohydrate binding activity (4). Since synthesis of the CSL in the presence of n,n'-dipyridyl prevented the post-translational modification of the lectin but did not prevent assembly into the high molecular weight complexes, it was possible to test this hypothesis directly.
H-4-11-E cells were labeled for 30 min with ["S]methionine and chased from 30 min in media containing unlabeled methionine. The pulse, as well as the chase, was performed in either the presence or absence of a,d-dipyridyl. Cell lysates were prepared by repeated freeze-thawing, brought to 25 mM CaC12, and applied to mannan-Sepharose columns equilibrated with wash buffer. After washing, the bound lectin was eluted with 25 mM. CSL in both the bound and unbound fraction was immunoprecipitated and analyzed by SDS-PAGE as shown in Fig. 3. Similar proportions of the CSL were found in the bound fraction after 30 min of chase in the presence and absence of a,d-dipyridyl. The CSL which was synthesized in the presence of a,d-dipyridyl and bound to the mannan-Sepharose had not been modified as evidenced by its lower M, when compared to CSL synthesized in the absence of n,a'-dipyridyl. Thus, neither hydroxylation of proline and lysine nor glycosylation of hydroxylysine are required for attainment of carbohydrate binding activity by the CSL. Complex assembly, rather than post-translational modification of the lectin, is required for expression of carbohydrate binding activity by the CSL. This suggests that complex assembly serves to form the binding site of the CSL.
Post  presence (a,a'-dipyridyl) or absence (untreated) of 3.5 mM @,a'-dipyridyl, H-4-11-E cells were labeled for 30 min with 50 pCi/ml [naslmethionine in methioninefree Earle's MEM in the presence or absence of the hydroxylation inhibitor. Cells were lysed in Triton lysis buffer, and cell supernatants were then brought to a final concentration of 25 mM CaCI2 prior to application to mannan-Sepharose. After sample application, 1-ml mannan-Sepharose columns were washed (4 X 1 ml) with wash buffer (20 mM Tris-HCI, pH 7.8, 25 mM CaC12, 100 mM NaCI, 0.05% Triton X-100) to remove unbound material (Unbound, 1-4). CSL MEM with (a,a'-dipyrdyl) or without (untreated) 3.5 mM a,&-dipyridyl, rat hepatoma cells were labeled with 50 pCi/ml ["Slmethionine in methionine-free Earle's MEM for 1 h and chased for 2, 6, and 18 h with Earle's MEM containing 5 mM unlabeled methionine. The hydroxylase inhibitor cup'-dipyridyl was present during pulse-labeling and chase periods in the lanes indicated (a,a'-dipyridyl). After media were removed cells were lysed with Triton lysis buffer. is the site where the lectin undergoes hydroxylation and glycosylation and is assembled into high molecular weight complexes, the time course for completion of these events is rapid when compared to the time course for secretion (3,4). Therefore, neither completion of post-translational modification nor assembly can account for retention within the Golgi. The impact of proline and lysine hydroxylation, as well as hydroxylysine glycosylation, on secretion of the CSL was examined as described above for attainment of carbohydrate binding activity. As illustrated in Fig. 4, in the presence of a,a'-dipyridyl virtually no CSL is detected in the medium of cells labeled for 1 h and chased for up to 18 h. Unmodified CSL was, however, detected within the cell a t each time. Densitometric analysis of fluorograms indicated that the amount of intracellular CSL present in cells treated with 3.5 mM a,a'-dipyridyl decreases only 15% between 2 and 18 h of chase. Thus, the absence of CSL in the medium of treated cells does not reflect rapid intra-or extracellular destruction of the unmodified form. Although the degradation of the CSL may be relatively more rapid in the absence of the posttranslational modifications, destruction would not seem to be able to account for its complete absence in the medium a t all times tested. Therefore, hydroxylation of proline and/or lysine and glycosylation of hydroxylysine plays an essential role in permitting or promoting the secretion of the CSL from the cell.
Post-translational Modifications of the CSL Are Required for Hydrophobic Interactions-Although the CSL is a soluble protein secreted by hepatocytes, both purification and stability are significantly improved by the presence of non-ionic detergents (l).' This suggested that the mature form of the CSL may interact with free lipid or membranes. T o determine if the CSL has hydrophobic domains capable of binding detergents, purified CSL was subjected to charge shift electrophoresis (22) in the presence of Triton X-100, deoxycholate, or CTAB (Fig. 5). In the presence of Triton X-100 alone, neither the CSL or GalBP, an integral membrane protein, migrated out of the sample well (data not shown). In the presence of 0.05% CTAB, a positively charged detergent, both the CSL and GalBP migrated to the negatively charged cathode, whereas in the presence of 0.25% deoxycholate, a negatively charged detergent, both the CSL and GalBP migrated to the positively charged anode. Thus, the CSL migrates according to the charge of the detergent rather than its inherent charge, indicating that the lectin binds these detergents.
Purified CSL is bound by agarose substituted with 10carbon alkyl chains and can be eluted with 2% Triton X-100.2 Interaction with such a hydrophobic matrix is consistent with the behavior of the CSL during charge shift electrophoresis and the stabilizing effect of non-ionic detergents and suggests that the CSL contains one or more hydrophobic "pockets" which interact with the alkyl chains conjugated to the agarose support (24). The CSL synthesized in the presence or absence of cu,a'-dipyridyl was subjected to hydrophobic chromatography in the presence of 25 mM CaC12 (Fig. 6) to determine if hydroxylation and subsequent glycosylation would influence the ability of the CSL to bind to hydrophobic matrices. Densitometric analysis of the fluorograms shown in Fig. 6 indicated that 88% of the CSL synthesized by control cells was bound by the agarose-Clo column and could be eluted with Triton X-100 (Fig. 6, Panel B, fractions 6-9). Binding was not calcium-dependent since subsequent elution with 2% Triton X-100, 2 mM EDTA did not remove any additional CSL (Fig. 6, Panel B, fraction 10). In contrast, only 18% of the CSL synthesized in the presence of a,d-dipyridyl was bound by the agarose-Clo column (Fig. 6, Panel D, fractions 1-5). Neither modified nor unmodified CSL appreciably bound underivatized agarose (Fig. 6, Panels A and C ) ; however, the small amount (5%) of the unmodified CSL which did bind this matrix was not eluted with Triton X-100 (Fig.   6, Panel C, fractions 6-9) but was eluted with 2% Triton X-100, 2 mM EDTA (Fig. 6, Panel C, fraction 10) suggesting that the unmodified form of the lectin may be able to interact weakly with agarose through its carbohydrate binding site. Thus, it would appear that hydroxylation of proline and lysine, and/or glycosylation of hydroxylysine results in the expression of hydrophobic binding by the lectin. It is possible that this property is required for efficient secretion from the cell into the medium.

DISCUSSION
The CSL is synthesized by hepatocytes and is found circulating in the blood. The kinetics of secretion for the CSL differ from the majority of other proteins synthesized and secreted by hepatocytes. The lectin appears in the extracellular medium as early as 2 h after synthesis and continues to be secreted at a constant rate over the next 6-10 h. The posttranslational processing of the CSL is complex. Proline and lysine residues in the lectin are hydroxylated, and the hydroxylysine formed is subsequently glycosylated to form Glc-GalHyLys have not directly demonstrated the relationship of individual alterations to the two discernible shifts in Mr. In addition to these modifications, the lectin is assembled into high molecular weight complexes from a low molecular weight form found predominantly in the endoplasmic reticulum. Hydroxylation, glycosylation, and assembly all appear to occur within the Golgi where the lectin is retained prior to secretion. The time course for these events is sufficiently rapid that processing of the CSL cannot account for the prolonged time required for secretion.
(2) previously determined that MBP-C contains hydroxyproline moieties within collagen-like sequences and suggested that hydroxylysine might also be present. Our demonstration that the CSL contains hydroxyproline, hydroxylysine, and glucosylgalactosylhydroxylysine and is assembled into high molecular weight complexes establishes that the lectin undergoes post-translational modifications which are characteristic of collagens and few other proteins. Other proteins known to contain collagen-like domains include the complement component Clq (6, 7), the asymmetric 18 S form of acetylcholinesterase (12-E), and pulmonary surfactant apoproteins (8)(9)(10)(11). The post-translational modifications of collagens and their impact on the assembly and physical-chemical properties of the collagens have been extensively studied (16)(17)(18). These studies indicate that hydroxylation of proline stabilizes the triple helical form of collagen and that lysine hydroxylation is important for intra-and intermolecular cross-links and fiber formation; however, the role of glycosylated hydroxylysine is less well established (16)(17)(18). A striking feature of the collagen, Clq, acetylcholinesterase, pulmonary surfactant apoproteins, and the CSL is that they all are assembled into high molecular weight complexes from subunits. This suggests that the collagenous domains of these molecules, and perhaps triple helix formation, may be involved in the assembly of these proteins into high molecular weight complexes.
In previous studies (4) we established that the post-translational modifications of the CSL and/or assembly into high molecular weight complexes are required to attain carbohydrate binding activity. Proline and lysine hydroxylation and hydroxylysine glycosylation account for the post-translational modifications previously detected as increases in M , and can Hydroxylation and glycosylation of the CSL are required for binding to hydrophobic matrices. Hydrophobic chromatography as described by Shaltiel (24) was used to determine whether the unmodified form of the CSL was able to interact with hydrophobic materials. H-4-11-E cells were labeled for 24 h with 50 pCi/ ml [3H]lysine in the presence (qd-dipyridyl) or absence (Control) of 3.5 mM a,d-dipyridyl. CSL which had been secreted into the media from the untreated cells and CSL which was released from the cr,a'-dipyridyl-treated cells by repeated freezing and thawing were brought to a final concentration of 25 mM CaC12 and applied to either agarose-Co (underivatized agarose) or agarose-Clo columns equilibrated with 20 mM Tris-HC1, pH 7.8,25 mM NaCl. Columns were washed with equilibration buffer (4 x 1 ml) and then eluted with equilibration buffer containing 2% Triton X-100. A final elution with 20 mM Tris-HC1, pH 7.8, 200 mM NaCl, 2 mM EDTA, 2% Triton X-100 (4 X 1 ml) was also performed to ensure removal of lectin from the agarose. Unbound lectin which included that which washed through the column upon loading and that which washed through with equilibration buffer (fractions 1-5) and the CSL which was eluted by either equilibration buffer containing 2% Triton X-100 (fractions 6-9) or 2% Triton X-100, 2 mM EDTA (fraction 10) were immunoprecipitated and analyzed by SDS-PAGE and fluorography. Arrows indicate where 2% Triton X-100 (Tr) elutions and EDTA ( E ) elutions begin. be blocked by the hydroxylase inhibitor ap'-dipyridyl (5). Assembly of the CSL into high molecular weight complexes and attainment of carbohydrate binding activity occurred normally when hydroxylation and glycosylation were prevented with a,a'-dipyridyl. Thus, it is assembly, rather than the post-translational modifications per se, which is required for expression of carbohydrate binding activity by the lectin. This is consistent with the observation by Drickamer et al.
(2) that the collagen-like domain can be proteolytically released and separated from the carbohydrate binding domain. In addition we have observed that the subunits of the CSL undergo interchain disulfide bond formation within 10-40 min after synthesis to form dimeric and trimeric forms. Interchain disulfide bond formation is sufficiently rapid to indicate it probably occurs prior to exit from the endoplasmic reticulum and prior to either of the lectin's post-translational increases in Mr. Disulfide bond formation and production of dimeric and trimeric species also proceeds normally in the presence of a,a'-dipyridyl, as would be expected for events occurring prior to the post-translational modifications being blocked.
A characteristic and unusual feature of the CSL is the prolonged time required for exit from the cell (3). The majority of this time is spent within the Golgi and follows completion of assembly, post-translational modification, and attainment of carbohydrate binding activity (4). In the absence of hydroxylation and glycosylation, the CSL is no longer secreted into the medium. The absence of lectin within the medium cannot be accounted for by intra-or extracellular degradation since recovery of lectin from within the cells is sufficient to account for the total amount of lectin synthesized even after prolonged incubations. This suggests that exit of the lectin from the cell under normal conditions is an active process and that this process cannot occur in the absence of hydroxylation and/or glycosylation. In addition, we have observed that the CSL binds detergent based on charge shift electrophoresis and that it is able to bind to hydrophobic matrices such as agarose-Clo and can be eluted with detergent. Thus, the CSL has the capacity to interact with hydrophobic domains of membranes or lipoproteins in the circulation. This property is lost if hydroxylation and/or glycosylation of the collagen-like domains is prevented, indicating that these posttranslational modifications have a major impact on the physical-chemical properties of the CSL and its biologic behavior. It is possible that the lectin must become associated with lipid prior to secretion and that this results in a prolonged sojourn within the Golgi. Notably, following secretion from hepatocytes some of the CSL can be found in the form of large complexes of 1.7-1.9 X lo6 Da which are disrupted by exposure to detergent, suggesting that they may represent a form of lipoprotein complex. ' We have not, however, been able to demonstrate CSL is present in lipoprotein fractions prepared from rat serum using KBr/NaCl gradients.
Like the CSL, the complement component Clq, asymmetric acetylcholinesterase, and the pulmonary surfactant apoproteins all contain collagen-like sequences, are assembled into high molecular weight complexes from disulfide-bonded units, and interact with lipid or membrane. The complement component Clq is the only other noncollagen protein thus far known to contain glycosylated hydroxylysine within collagenlike sequences (6). In its mature form Clq consists of three polypeptide chains (A, B, C), each containing a collagenous domain, which are disulfide-linked into dimers and assembled into a complex of 18 subunits which is a complex of six triple helical structures (6, 7). The post-translational processing and assembly of these subunits into the mature form has not yet been examined. However, it has been demonstrated that the secretion of C l q like that of the CSL and interstitial collagen is inhibited by the hydroxylase inhibitor a,a'-dipyridyl (7,27,28). Hydroxylation and subsequent glycosylation of the hydroxylysine residues of C l q also appear to be necessary for its secretion. In addition, Loos (7) suggests that the lag time observed in the secretion of Clq after synthesis may result from a temporary interaction with the membrane via its collagenous domains. After secretion and assembly, the collagenous regions of Clq are believed to provide the binding site for the (Clr,-Cls,) complex (7).
Asymmetric acetylcholinesterase interacts with membranes through collagenous sequences. The asymmetric acetylcholinesterase consists of 12 subunits (Al2) linked by disulfide bonds to three collagen-like tail polypeptides (12)(13)(14)(15). Like the 150 to 260-kDa form of the CSL, this AI, form of acetylcholinesterase is assembled and retained in the Golgi prior to transport to the cell surface (29). The collagen-like tails of the acetylcholinesterase have been shown to be required for association of acetylcholinesterase with membranes, because digestion with collagenase disrupts this membrane interaction (12).
The pulmonary surfactant proteins also contain collagenous domains and interact with lipid. In uiuo, pulmonary surfactant apoproteins are found in large complexes with lipids and serve to alter the physiochemical characteristics of lung surfactant by increasing spreading of the lipid monolayer and adsorption to air/liquid interfaces (30)(31)(32)38). These proteins are remarkedly similar to the CSL. Drickamer et al.
(2) demonstrated that MBP-C (or CSL) and canine pulmonary surfactant apoprotein (10) show extensive sequence homology and possess similarly localized collagenous domains a t their amino termini. The predominant forms of the surfactant apoproteins consist of a 26-kDa unglycosylated subunit and two sialylglycoprotein subunits of 32-36 kDa which are assembled into high molecular weight disulfide-bonded complexes (9-11, 31, 33-37). Pulmonary surfactant apoproteins are retained within cultured rat type I1 epithelial cells prior to sialylation and are subsequently secreted with the same time course as pulmonary lipids (37). This suggests that lipid and the pulmonary surfactant apoproteins are assembled prior to secretion. Recently Ross et al. (38) presented evidence which indicates that amphiphilic amino acids between residues 81 and 117 and the amino-terminal collagenous domain both contribute to canine pulmonary surfactant protein interaction with phospholipid.
The properties of pulmonary surfactant proteins and those of the CSL are similar in a number of respects. Binding to detergent and hydrophobic matrices by the CSL is dependent on hydroxylation and glycosylation of the collagen-like region but is not Ca2+-dependent. The ability of the CSL and the other proteins containing collagenous domains to interact with hydrophobic moieties may actually reflect triple helix formation, which requires hydroxylation and hydroxylysine glycosylation. The triple helical domains may directly interact with lipid and/or membranes or influence the conformation of another domain which actually participates in the interaction. Thus, the amino-terminal collagen-like region of the CSL may participate in its interaction with lipids and/or membranes in the Golgi in a manner analogous to that of the surfactant proteins. This interaction could serve to retain the CSL within the Golgi and to ultimately allow its transport as a lipid-protein complex out of the cell. No carbohydratespecific binding activity has thus far been detected for the surfactant protein; however, the extensive homology with the CSL suggests that there may be an independent carbohydrate binding site on surfactant protein.
Although collagen pro-a chains and the CSL undergo several similar intracellular modifications, the subcellular location proposed for these events for collagen differs from that which we have found for the CSL. During and/or shortly after synthesis, collagen pro-a chains are hydroxylated by prolyl and lysyl hydroxylases to produce 4-hydroxyproline, 3-hydroxyproline, and hydroxylysine (16)(17)(18). Based on the kinetics of hydroxylation and glycosylation, and the subcellular localization of the hydroxylase and glycosyltransferase enzymes these modifications of collagen are proposed to occur within the rough endoplasmic reticulum and proceed until the collagen triple helix is formed (39)(40)(41)(42)(43)(44). In contrast, the posttranslational modifications of the CSL appear to occur within the Golgi (4). This raises the possibility that hydroxylation and glycosylation of proline and lysine in collagen occur at a different site than for the collagen-like sequences of the CSL. Alternatively, some or all of these post-translational modifications of collagen may occur in the Golgi rather than the endoplasmic reticulum as previously thought. Resolution of this issue will require re-examination of collagen synthesis by hepatoma and other cell types.