OF CHEMISTRY No. of November 25, pp. 1985 by Inc U.S.A ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 27, Issue of November 25, pp. 14732-14742,1985 Printed in U.S.A.

The Biosynthesis, Processing, and Secretion of Laminin by Human Choriocarcinoma Cells*

(Received for publication, June 5, 1985)

Barry P. Peters, Richard J. Hartle, Raymond F. Krzesicki, Todd G. Kroll, Fulvio Perini, James E. Balun$, Irwin J. Goldstein$, and Raymond W. Ruddon From the DeDartments of Phnrmacolom and tBioloeical Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109 ’

-., . -~

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (M, -400,000) and B (M, = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of a-mannosidase I since the principal laminin forms that accumulate in JAR cells contain ManeGlcNAc2 and MansGlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (M, -950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one- third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

We have observed that a human malignant cell line (JAR) derived from choriocarcinoma synthesizes and secretes laminin, a glycoprotein component of the basal lamina. First

* This investigation was supported by United States Public Health Service Grants CA-32949 and CA-20424 awarded by the National Cancer Institute, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

isolated from the extracellular matrix of murine tumors (1- 4), murine laminin is composed of two distinct polypeptide subunits, A (Mr = 400,000-440,000) and B (Mr = 200,000- 220,000), linked by disulfide bonds (1, 5-7) in a cruciform structure (5-7).l Antibodies against mouse tumor laminin have been used to localize immunoreactive laminin in the thin continuous extracellular sheets of basal lamina that separate the epithelial and endothelial cell layers from the connective tissue stroma (1, 2 , 8-11). Laminin appears to function as an adhesive molecule in that different domains of the laminin molecule are capable of interacting noncovalently either with the cell surface (7, 12), perhaps via a specific laminin receptor (13-17) or sulfated glycolipid (18), and with the proteoglycan and collagenous elements of the extracellular matrix (7, 19-22). The biosynthesis of laminin subunits has been observed in cultured murine (2,23-34) and human (35- 38) cells.

Morphologically aberrant basal laminae have been fre- quently observed in human solid tumors of epithelial origin. Rather than the thin, continuous structures found in healthy tissue, basal laminae separating islands or cords of malignant epithelial cells from the connective tissue stroma may be discontinuous, reduplicated, or entirely absent (39-47). Since the intact basal lamina has been regarded as a barrier to the invasive or metastatic spread of malignant cells (35, 48, 49), the loss of this structure could be crucial to tumor progression. For example, the metastatic potential of certain murine tumors has been correlated with the secretion of lytic enzymes capable of degrading basal lamina components (50-53). Since epithelial cells are the biosynthetic source of the basal lamina to which they are anchored (54), another hypothesis is that some malignant cells may be incapable of biosynthesizing a normal basal lamina.

As a first step in testing this hypothesis, we have charac- terized the biosynthesis, subunit assembly, post-translational modification, and secretion of laminin by a malignant cell line (JAR) established from human gestational choriocarcinoma. Our results clearly establish JAR as a laminin-produc- ing tumor cell line that should prove to be a source for the isolation of human laminin. Furthermore, our previous studies on the biosynthesis of the placental glycoprotein hormone chorionic gonadotropin by JAR cells (55-58) enable us to

Three nomenclatures have been used in the literature to describe the laminin subunits. Rao et al. (7) use and a, Carlin et al. (32) use GP1 and GP2 while Kurkinen et al. (67) use A and B to designate the larger (Mr = 400,000) and smaller (M, = 200,000) laminin subunits, respectively. We have chosen to use the designation of Kurki- nen et al. for laminin in order to avoid confusion when we compare the biosynthesis of the laminin subunits (A and B) and the chorionic gonadotropin subunits (a and 0 ) that are synthesized in the same cell.

14732

Laminin Synthesis by Human Choriocarcinoma Cells 14733

compare the biosynthesis and secretion of the matrix glycoprotein laminin and the secretory glycoprotein hCG2 in the same cells.

EXPERIMENTAL PROCEDURES3

Biosynthetic Labeling of Cells-JAR choriocarcinoma cells obtained from Dr. Roland Pattillo, Medical College of Wisconsin (591, were grown in Dulbecco’s modified Eagle’s medium containing D-glUCOSe (4500 mg/liter) and supplemented with 10% fetal bovine serum (both from Gibco). Cells grown to late log stage (approximately 80% of confluency) in 100-mm plastic dishes (2 x lo7 cells/dish) were biosynthetically labeled with either ~-[~~S]methionine (1100 Ci/mmol, New England Nuclear) or with D-[2-3H]mannose (14 Ci/mmol, New England Nuclear) at 37 “C in an atmosphere of 95% air, 5% CO,. Labeling with [35S]methionine (150 pCi/ml) was carried out for either 10 or 60 min, as indicated, in serum-free, methionine-free growth medium (5 ml/dish) after a 30-min “starvation” in the methionine- free medium. Labeling with [3H]mannose was carried out either in serum-free growth medium (5 ml/dish, 1 mCi/ml) for 10 min or in complete growth medium (5 ml/dish, 140 pCi/ml) for 20 h. In some experiments, the labeling medium was removed, and the cells were “chased” with complete growth medium for intervals up to 8 h.

Preparation of Cell Lysates, Immunoprecipitation, and SDS- PAGE-JAR cells were lysed in PBS buffer containing detergents (Triton X-100, sodium deoxycholate, and SDS) and the protease inhibitors phenylmethanesulfonyl fluoride (2 mM), N-ethylmaleimide (10 mM), and EDTA (20 mM) as previously described (55). The detergent/protease inhibitors mixture was also added to the harvested culture media as a &fold concentrated solution. In some experiments, additional protease inhibitors (leupeptin, antipain, benzamidine, Tra- sylol, chymostatin, and pepstatin) were added to the lysis buffer, as employed by Ronnett et al. (61) in studies on the insulin receptor. The laminin forms were immunoprecipitated from the detergent lysates with rabbit antiserum against mouse EHS tumor laminin (60) (1 p1/5 ml of lysate) and Protein A-Sepharose beads (Pharmacia, 100 pl of packed beads/pl of antiserum). The partial cross-reactivity of polyclonal anti-murine laminin antibody with human laminin has previously been shown by Rohde et al. (62). The immunoadsorbed laminin was eluted from the washed Protein A-Sepharose pellets with an equal volume of boiling 2-fold concentrated SDS sample buffer either with or without 2% 2-mercaptoethanol as indicated. The laminin polypeptides were then fractionated by SDS-PAGE on polyacrylamide gradient slab gels (3-10% with 3% stacking gel, unless otherwise indicated) in the Laemmli buffer system (63). The slabs were impregnated with ENHANCE (New England Nuclear) in order to visualize the radioactive bands by fluorography of the dried gels. Nonradioactive proteins were stained by the silver technique of Merril (64).

Two-dimensional SDS-PAGE analysis of the laminin immunoprecipitate was carried out in the Laemmli system without reduction of disulfide bonds prior to electrophoresis in the first dimension and with reduction of disulfide bonds prior to electrophoresis in the second dimension. Electrophoresis in the first dimension was performed on tube gels (0.2 cm X 12 cm) of 4% polyacrylamide without a stacking gel. The laminin immunoprecipitate was boiled for 5 min in 2-fold concentrated sample buffer lacking 2-mercaptoethanol and applied directly to the top of the running gel. The tube gel was subjected to 400 V for 50 min and then 500 V for an additional 20 min, extruded, and soaked for 20 min at ambient temperature on a rocker platform in 5 ml of pH 6.8 stacking gel buffer containing 2% 2-mercaptoetha-

The abbreviations used are: hCG, human chorionic gonadotropin; endo H, endo-@-hexosaminidase H (EC 3.2.1.96); PBS, phosphate- buffered (pH 7.4) saline (0.15 M sodium chloride, 0.01 M sodium phosphate); DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

Portions of this paper (including part of “Experimental Proce- dures,” part of “Results,” and Figs. 2-6 and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 85M-1852, cite the au- thors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

nol. The tube gel was then applied to the stacking gel (3% polyacrylamide) of a 3-10% polyacrylamide gradient slab with an overlayer of agarose (1%) in pH 6.8 stacking gel buffer. The slab gel was run at 65 V, and the radioactive bands were located by fluorography in the usual manner.

Pulse-Chase Kinetic Analysis of Laminin Biosynthesis, N-linked Oligosaccharide Processing, and Secretion-JAR cells were pulsed for 10 min with either [35S]methionine or [3H]mannose and then chased for intervals up to 8 h. The immunoreactive laminin forms were precipitated from the cell lysates and chase media and fractionated by SDS-PAGE. The laminin forms described in this report, and the hCG forms described in a previous publication (58) were immunoprecipitated sequentially from the same cell lysates and chase media. The [35S]methionine-labeled bands were excised from the dried SDS gel using the fluorography film as a template. The polyacrylamide slices were depolymerized with 30% hydrogen peroxide and counted in the liquid scintillation spectrometer. The [3H]mannose-labeled bands were cut out of the dried SDS gels and eluted from the slices as glycopeptides with protease treatment. These methods have been described elsewhere (58, 65). The laminin glycopeptides were then digested with endo H (65), and the array of endo H-sensitive high mannose oligosaccharides and endo H-resistant complex glycopeptides were analyzed by gel filtration chromatography on a column of Bio-Gel P-4 (57) or by the following HPLC protocol (65). Aliquots of the endo H digestion mixtures were adjusted to a volume of 350 pl with the endo H-citrate buffer (50 mM sodium citrate, pH 5.6) mixed with 650 pl of acetonitrile and injected onto an Altex carbohydrate column (4.1 mm X 30 cm) equilibrated in 65% acetonitrile/water. The column was eluted at a flow rate of 2 ml/min with a linear gradient decreasing to 50% acetonitrile over 25 min, increasing to 65% acetonitrile over 3 min, then isocractic at 65% acetonitrile for 18 min. Fractions of 0.5 min (1.0 ml) were collected and counted in 5 ml of Atomlight scintillation fluid (New England Nuclear). The column was calibrated with a series of tritiated high mannose oligosaccharides ((Man),GlcNAc, 9 2 n 2 5) available in our laboratory from previous studies (57). The recovery of the neutral oligosaccharides from the column was 80-90%. A representative HPLC elution profile is shown in Fig. 4 of Ref. 58.

Digestion of Cell-associated Laminin with Trypsin-In order to distinguish extracellular (trypsin-sensitive) laminin from intracellular (trypsin-resistant) laminin, JAR cells were labeled continuously with [3H]mannose for 20 h. The washed cell layers were treated with trypsin-EDTA (Gibco) according to the same protocol routinely used to subculture the cells, except that the incubation time was 20 min rather than 10 min in order to achieve a single-cell suspension. The trypsin-treated cell suspension was diluted into an equal volume of serum-containing culture medium, the cells were collected by centrifugation and lysed in 5 ml of detergent buffer, and laminin was immunoprecipitated. The laminin from the trypsin-treated cells was compared by SDS-PAGE to laminin from a control culture. A portion of each of these immunoprecipitates was also digested with protease and then endo H, and the [3H]mannose-labeled oligosaccharides were analyzed by gel filtration on the Bio-Gel P-4 column.

RESULTS

Laminin Subunits Produced by Human Choriocarcinoma Cells-JAR cells were pulse-labeled for 10 min with [35S] methionine and then chased with nonradioactive culture medium for intervals ranging from 0 to 8 h. The immunoreactive laminin forms were immunoprecipitated from the cell lysates and chase media and examined by SDS-PAGE (Fig. 1). After reduction of the immunoprecipitated proteins with 2-mercaptoethanol, labeled polypeptides were observed to migrate on the SDS gel at two loci, designated by the brackets in Fig. lA , corresponding in apparent molecular weight to the A (Mr -400,000) and B (Mr = 200,000) subunits of laminin. The molecular weight of the human laminin polypeptides was estimated by comparison to subunits of laminin isolated from the EHS tumor (Fig. 2, Miniprint Section) and to rabbit muscle myosin (Mr = 200,000). The specificity of the laminin immunoprecipitation was shown 1) by substituting nonim- mune serum for anti-laminin serum (Fig. 3, Miniprint Sec- tion), and 2) by adding excess unlabeled EHS laminin to JAR

14734 Laminin Synthesis by H u m a n Choriocarcinoma Cells A . Laminin (reduced)

Lam - B. Laminin (non-reduced)

Chase time: 0 7.5 1530 1 2 4 8 7.51530 1 2 4 8 ""

min hrs mln hrs

C e l l s M e d i a -

FIG. 1. Pulse-chase labeling of laminin synthesized by human choriocarcinoma cells. JAR cultures (2 X lo7 cells in 100- mm dishes) were pulsed for 10 min with [35S]methionine and chased for intervals ranging from 0 to 480 min. The laminin forms were immunoprecipitated from cell lysates and chase media. Half of each immunoprecipitate was reduced with 2-mercaptoethanol (A) and half was not reduced ( B ) prior to analysis by SDS-PAGE on a 3-10% polyacrylamide gradient slab gel with a 3% acrylamide stacking gel. Radioactive bands were visualized by fluorography (5-day exposure). The stacking gel remained intact in the fluorograph of B. In A, the laminin subunit precursor forms (PA, pB) are indicated by the arrowheads marked +; mature laminin subunits (A, B) are indicated by arrowheads marked 0. In B, the assembled laminin molecule is designated Lam; the uncombined B and dimer B forms are designated p B and pBlpBz, respectively.

culture fluids to block competitively the immunoprecipitation of labeled laminin forms (Fig. 4, Miniprint Section). Further- more, anti-fibronectin (Fig. 3, Miniprint Section) and anti- EHS type IV collagen (data not shown) failed to precipitate labeled polypeptides from the JAR cell lysates.

Post-translational Processing and Secretion of Laminin- The laminin A and B subunits that first appeared in the cells during the biosynthetic pulse were processed during the chase incubation to forms that migrated differently on SDS-polyacrylamide gels. For example, a single A subunit precursor that first appeared in the cell lysate after the 10-min pulse (PA, designated in Fig. lA by the arrowhead marked +) gave rise to a pair of mature A forms (A and A', designated by the arrowheads marked o), one migrating more slowly (A) and one migrating more rapidly (A') than the precursor from which it was formed. The existence of three distinct A subunit forms (A, PA, and A') may be more clearly seen in the SDS gels shown in Figs. 2 and 6 (Miniprint Section). The mature A forms were visible in the cell lysates as early as 30 min of chase and began to appear in the chase medium in 1 h. The production of the smaller mature A' subunit seemed to be closely timed with the secretion of the laminin molecule, since A' was a minor component of cell-associated laminin but was a major component of secreted laminin (Fig. lA, compare 8- h chase cells with 4- or 8-h chase media). The ratio of A to A' in secreted laminin did not change significantly with time of chase (Fig. lA, compare 4- and 8-h chase media). Inclusion of the expanded panel of protease inhibitors in the lysis

buffers (see "Experimental Procedures") failed to prevent the appearance of A' (data not shown).

A pair of B subunit precursors (pB1 and pB2, designated by the arrowheads marked +) gave rise to a pair of more slowly migrating mature B forms (B1 and B2, designated by the arrowheads marked 0). The mature B forms were visible in the cell lysates and appeared in the medium by 2 h of chase. The doublet of mature B can be clearly seen in the chase media (Fig. lA, 4- and 8-h chase media; Figs. 2 and 6, Mini- print Section). However, in cell lysates, the mature B doublet is partially obscured because it overlaps the pB doublet, such that three rather than four bands are seen at the B locus (Fig. lA, 8-h chase cells). Our claim that cell lysates indeed contain a pair of mature B forms is based on the observation of four distinct B subunit bands (pB,, pB,, B1, and B,) in 4-h chase cell lysates after the immunoprecipitates are treated with endo H to increase the mobility of the pB precursor forms on SDS- PAGE (Fig. 5, lane 12, Miniprint Section).

The A subunit is processed to maturity more rapidly than the B subunit forms. Maturation is half-complete by 1 h of chase for A and by 4 h of chase for B. The conversion of the laminin A and B subunit precursors to mature forms involves the processing of their asparagine-linked carbohydrate chains from high mannose to complex units. This was established by the observations that the laminin precursors migrated more rapidly on SDS-PAGE after digestion with endo H but were unaffected by sialidase whereas the mature forms were unaffected by endo H but migrated more rapidly on SDS-PAGE after sialidase treatment (Figs. 5 and 6, Miniprint Section). Furthermore, tunicamycin blocked the addition of the endo H-sensitive oligosaccharides to the newly synthesized laminin subunits (Fig. 5, Miniprint Section). Based on these experiments, we know that the laminin A and B subunits secreted into the culture medium during the 8 h of chase incubation (Fig. lA) are the endo H-resistant, sialidase-sensitive mature forms (A, A', B,, and B,) (Figs. 5 and 6, Miniprint Section).

Assembly of the Laminin Molecule-The extent of laminin subunit combination in cell lysates and culture media was assessed by SDS-PAGE in the absence of 2-mercaptoethanol. Under these conditions, the disulfide bonds joining the laminin subunits remain intact so that the assembled laminin molecule may be fractionated on SDS gels from uncombined A and B subunits. In addition to M, -400,000 and M, = 200,000 forms, the nonreduced cellular immunoprecipitates also contained a larger form that penetrated the SDS-polyacrylamide gel by only 3-4 mm (designated Lam in Fig. 1B) and co-migrated with undissociated EHS laminin (Mr -950,000) (Fig. 2, Miniprint Section). The appearance of assembled laminin in the cell lysates at the earliest chase time (Fig. lB, 0-min chase cells) indicates rapid combination of the newly synthesized laminin subunits.

Further evidence that the M, -950,000 form in JAR cell lysates is the fully assembled laminin molecule is provided by two-dimensional SDS-PAGE, without reduction of disulfide bonds prior to electrophoresis in the first dimension and with reduction of disulfide bonds prior to electrophoresis in the second dimension (Fig. 7). The M, -950,000 form dissociated upon reduction into a mixture of A and B subunits. The pA subunit was more heavily labeled with [35S]methionine than the doublet pB subunit forms. Both pB1 and pB2 subunits participated in the formation of laminin as seen by the equal intensity of the two pB spots derived from the intact molecule (Fig. 7).

We originally suspected that the M, -400,000 and M, = 200,000 laminin forms in the cell lysates, apparent on the nonreduced SDS gel in Fig. lB, corresponded to uncombined


1 . NON-REDUCED 0

400K 950 K 1 1

* 01

0 0

P

rn 3

0 c 0

0 rn

PA Lam + PBIPB,

FIG. 7. Two-dimensional SDS-PACE analysis of the laminin forms in J A R cell lysate. Laminin immunoprecipitated from the cell lysate after a 1-h biosynthetic pulse with ['5SJmethionine was subjected to the two-dimensional SDS-PACE as described under "Experimental Procedures." Electrophoresis in the first dimension was performed on a tube gel without reduction of disulfide bonds; electrophoresis in the second dimension was performed by applying the tube gel, infused with 2-mercaptoethanol, to the top of the polyacrylamide slab. The open circle denotes the origin of the two- dimensional gel. A duplicate nonreduced tube gel (denoted by the hmcket) dried alongside the two-dimensional gel, shows the migration of the M. -400,000 and M . -950,000 laminin forms in the first dimension. The laminin subunit precursors are designated PA, p R I , and pR,; pRlpH2 is the disulfide-linked R dimer; Lam is the assembled laminin molecule: and A is the mature A subunit.

A and B subunits, respectively. It can be seen from the two- dimensional SDS gel in Fig. 7, however, that most of the M , -400,000 form dissociated in the presence of 2-mercaptoethanol to yield a doublet a t M , = 200,000, suggesting that it is actually a disulfide-linked dimer of the B subunit (designated pBlpR, in Fig. 1R and Fig. 7). Only a trace of the M, -400,000 form was unaffected in molecular weight by reduction of disulfide bonds, representing a small intracellular pool of uncombined A subunit. Both B subunit forms (pBl and pB2) participated in B dimer formation, as shown by the equal intensity of the B, and B2 spots derived from the M , -400,000 molecule (Fig. 7). This suggests a pBlpBn heterodimer structure, although we cannot rule out the possibility that pBIpBl and pB2pRn homodimers also occur. The M, = 200,000 form on the nonreduced gel in Fig. 1E is most likely uncombined pB subunit. Only 1 pB band was observed, corresponding in mobility to pB2, although nonreduced pBl and pB2 may co- migrate on the SDS gel.

The reduced SDS gel (Fig. 1A) clearly showed the matura- t.ion of the cellular laminin forms, but these processing events seem to be restricted to the assembled laminin molecule since uncombined pB and pBlpBn dimer were not converted to the more slowly migrating mature counterparts during the chase (Fig. 1R). Furthermore, the laminin secreted or shed by JAR cells into the culture medium was predominantly the fully assembled molecule. Only traces of partially assembled lami-

nin forms were observed in the chase medium. Therefore, in JAR cells, the assembly of the laminin molecule appears to be a prerequisite for both the maturation of its subunits and for secretion.

Kinetics of Laminin Biosynthesis, Subunit Combination, and Secretion-In order to quantitatively analyze the pulse-chase kinetic data, the laminin bands were excised from the SDS gels in Fig. 1, and the polyacrylamide slices were depolymerized in hydrogen peroxide and counted (Fig. 8, A and R). Taking as 100% the sum of [:"S]methionine in all immunoreactive laminin forms in the cell lysates at 30 min of chase (the point of maximum ['"Slmethionine incorporation), the nonreduced gel (Fig. 8A) showed that after 8 h of chase, 52% of the incorporated label was recovered as the assembled cell- associated laminin molecule, 11% was cellular uncombined pB and pBlpB2 dimer forms, and 20% was laminin secreted into the chase medium, for an overall recovery of 8.3%. The reduced SDS gel (Fig. 8R) showed that at 8 h of chase, 66% of the B subunit was associated with the cells while 24% had been secreted, for an overall B recovery of 90%. At the same chase time, 48% of the A subunit was associated with the cells while 23% had been secreted for an overall recovery of 7lrE for the A subunit.

The nonreduced gel (Fig. 8A) revealed a kinetic precursor- product relationship between the uncombined pR and pRlpB, dimer forms and the assembled laminin molecule in that the pools of incompletely assembled subunits had achieved a plateau of maximum labeling at 7.5 min of chase and then declined after 30 min, whereas the pool of assembled laminin accumulated label up to 30 min of chase. Thereafter, the cellular laminin pool remained constant for the duration of the chase as the loss due to secretion was exactly counterbal- anced by the continuing assembly of laminin from the labeled pools of pB and pBlpRn forms that persisted in the cells and from newly synthesized unlabeled PA. The first order half- time for secretion of the laminin molecule was 100 min as calculated from the slope of the semilog transformation of the secretion curve (58).

Kinetics of Processing of the N-linked Oligosaccharides of Laminin-The same pulse-chase protocol as described above for ["S]methionine was repeated with ["Hjmannose in order to determine the kinetics of processing of the N-linked glycans of the A and B laminin subunits and to pinpoint the rate- limiting step in the process. The laminin immunoprecipitates were fractionated by SDS-PAGE after reduction with 2-mercaptoethanol (gel not shown). and the glycoprotein bands were excised and eluted from the gel slices as glycopeptides by treatment with protease. The glycopeptides were treated with endo H, and the released oligosaccharides were fractionated by HPLC.

The total 'H cpm in the protease eluates of the laminin A and B subunit bands are plotted as a function of chase time in Fig. 8C. The ['Hlmannose experiment corroborated the ["S]methionine experiment with regard to the kinetics of laminin synthesis and secretion (compare Fig. 8, R uersus C) except that, as we also observed for hCG (58), the ['HI mannose labeling lagged behind the [:"S]methionine labeling by about 30 min.

The 'H cpm contained in the different high mannose oligosaccharide fractions is plotted as a function of chase time in Fig. 9. The newly synthesized laminin pA and pR precursors that appeared in the cells between 0 and 30 min of chase contained principally Man,GlcNAc, oligosaccharide units. Only a t race of (hexose),,,GIcNAc, (presumably Glc- Man9GlcNAc2) was found in the A glycopeptides, and none in the B glycopeptides. indicating the rapid removal of the

14736 Laminin Synthesis by Human Choriocarcinoma Cells

A. Laminin (non-reduced) Lam (cells)

0

B. Laminin (reduced)

C. Laminin (reduced) p\

10,000

z 0 I

5,000

0 60 120 240 400

CHASE TIME (MIN.)

FIG. 8. Pulse-chase kinetics of the biosynthesis and secretion of laminin by human choriocarcinoma cells. JAR cultures were pulsed for 10 min with [35S]methionine (A and B ) or [3H] mannose (C) and chased for intervals of 0, 7.5, 15, 30, 60, 120, 240, and 480 min, and the laminin forms were immunoprecipitated from the cell lysates and chase media. The [35S]methionine-labeled immunoprecipitates were divided in half for SDS-PAGE with and without 2-mercaptoethanol in the SDS sample buffer. Fluorographs of these gels are shown in Fig. 1. One-third of the [3H]mannose immunoprecipitates was taken for SDS-PAGE after reduction with 2-mercaptoethanol (gel not shown). The radioactive bands were excised from the gels using the exposed fluorography film as a template. From the nonreduced gel (A) , intact laminin (Lam), the disulfide- linked pBlpBp dimer, and uncombined pB subunit bands were sliced and counted. From the reduced gels ( B and C), the A and B subunit loci were sliced as indicated by the brackets in Fig. lA so as to contain the sum total of the A (PA + A + A') and B (pB1 + pB2 + B1 + B,) polypeptides. The [35S]methionine-labeled bands were counted after depolymerizing the polyacrylamide in hydrogen peroxide. The [3H] mannose-labeled bands were counted after they were eluted from the gel slices with protease. The radioactivity in the cell-associated laminin forms is represented by circles; secreted laminin is represented by triangles. A, [35S]methionine-labeled laminin, nonreduced; B, [35S] methionine-labeled laminin, reduced; C, [3H]mannose-labeled laminin, reduced.

V

r CY 10,000 -

B. Laminin B

.A< - .-(3

C h a s e l i m e ( m i " . )

/. .. -0""""-

"""""_ a

7.5 60 120 240 480

Chase time (rnin.)

FIG. 9. Kinetics of processing of the asparagine-linked oligosaccharides of the cellular laminin precursors. The content of high mannose oligosaccharides in the cellular laminin immunoprecipitates was determined at different chase times of 0, 7.5, 15, 30, 60, 120, 240, and 480 min following a 10-min pulse with [3H]mannose. The laminin A and B subunit loci were excised from the SDS gel described in Fig. 8C and eluted as glycopeptides with protease. The high [3H]mannose oligosaccharide content in the glycopeptide eluates was determined by HPLC after digestion with endo H. The total amount of tritium in each oligosaccharide peak is plotted as a function of the chase time. Inset to B, the decay of total endo H-sensitive [3H] mannose from the cellular laminin A (A) and laminin B ( B ) immunoprecipitates is shown as a function of chase time, taking as 100% the respective values at 60 min of chase. A, laminin A subunit; B, laminin B subunit. GMJi, GlcMangGlcNAc; MgN, MangGlcNAc; M a , MaQGlcNAc; M5N+M&+M7N, Man5GlcNAc + Man6GlcNAc + Man7GlcNAc.

peripheral glucosyl units. The rate-limiting step in the oligosaccharide processing of laminin was the trimming of the MangGlcNAcz units, a reaction catalyzed by a-mannosidase I (66), as shown by the persistence during the chase of cellular laminin precursor forms containing predominantly MangGlcNAcz and MansGlcNAcz oligosaccharides. The laminin B subunit appeared to be a better substrate for the mannose trimming reaction as shown by the more rapid appearance of MansGlcNAcz and smaller high mannose units in this subunit during the chase. For example, as an index of the rate of trimming, by 60 min of chase the ratio of counts in MansGlcNAcz to MangGlcNAc2 was 0.78 for B compared to 0.40 for A. Virtually identical results were obtained in a second experiment (data not shown) in that the same ratio was 0.72 for B compared to 0.43 for A. Interestingly, even though the initial rate of trimming of the high mannose chains appears to be slower, the A subunit was processed to endo H resistance more rapidly than the B subunit. The more rapid maturation of pA compared to pB forms can be seen in Fig. lA and is substantiated by the more rapid decay of endo-H- sensitive [3H]mannose from the A subunit (inset to Fig. 9B). The persistence of endo H-sensitive oligosaccharides corresponding to 40-50% of the incorporated [3H]mannose in the cellular laminin A and B subunits at 8 h of chase suggests


that some of the N-linked glycans of laminin remain un- processed beyond the high mannose stage even in the mature laminin molecule.

Mature Cell-associated Laminin Forms Are Located on the Cell Surface Whereas the Laminin Precursors Are Intracellular Forms-Immunoreactive laminin was immunoprecipitated from JAR cell lysates and labeling media after a continuous 20-h incubation with [3H]mannose. Half of each immunoprecipitate was analyzed by SDS-PAGE (gels not shown), while the remaining half was analyzed for its content of [3H]mannose-labeled oligosaccharides after digestion with protease and endo H. Oligosaccharides liberated by endo H were fractionated from endo H-resistant glycopeptides by gel filtration on a calibrated column of Bio-Gel P-4 (Fig. 10). Some of the labeled cells were treated with trypsin before the cell lysates were prepared in order to discriminate between intracellular laminin, shielded from the trypsin, and cell surface laminin, exposed to the trypsin.

The cell-associated laminin was a mixture of precursor and mature forms that contained [3H]mannose-labeled oligosaccharides divided equally between endo H-sensitive and endo H-resistant structures (Fig. 1OA). The predominant endo H- sensitive oligosaccharides of the cell-associated laminin precursors were Man9GlcNAc2 and Man8GlcNAcz, consistent with the slow rate at which those oligosaccharides are processed in JAR cells (Fig. 9). The secreted laminin was composed of mature subunits that contained 70% of the [3H] mannose in endo H-resistant oligosaccharides and 30% in high mannose oligosaccharides (Fig. 1OC).

Trypsin treatment of the [3H]mannose-labeled cells had no effect on the cellular content of the laminin precursor forms, as seen by the unperturbed array of high mannose oligosaccharides in the immunoprecipitates (Fig. 10B). However, the cell-associated mature forms containing complex oligosaccharides were lost. These results indicate that the laminin precursors are intracellular, presecretory forms protected from the proteolytic action of the trypsin, whereas the mature cell- associated laminin has been externalized by the cell in a trypsin-sensitive location. The negligible amount of trypsin- resistant, endo H-resistant laminin associated with the cells shows that mature laminin does not accumulate intracellularly but is rapidly externalized by the cell upon completion. Immunohistochemical staining of JAR cultures with anti- laminin serum provides additional evidence that immunoreactive laminin is expressed in punctate clusters on the surface of viable cells (Fig. 11, Miniprint Section).

DISCUSSION

Characteristics of Human Laminin Subunits Synthesized by Choriocarcinoma Cells in Culture-JAR cells synthesize and secrete (or shed) into the culture medium a glycoprotein that is immunologically cross-reactive with murine laminin from the EHS sarcoma. The human laminin is composed of the characteristic A (M, -400,000) and B (Mr = 200,000) subunits (1,5, 7) linked by disulfide bonds to form a molecule with the same size on nonreducing SDS gels as the EHS laminin molecule (Mr -950,000) (Fig. 2). Like murine laminin (25, 32, 67), both the A and B subunit forms of JAR laminin are initially glycosylated with high mannose asparagine-linked carbohydrate units, as demonstrated by the effects of tunicamycin and endo H on the newly synthesized laminin precursors (Fig. 5, Miniprint Section) that appear in the cells immediately following a 10-min biosynthetic pulse with [35S] methionine or [3H]mannose. During 8 h of chase incubation, the precursors are converted to the more slowly migrating endo H-resistant mature forms, a process that entails the

I [L 0 I

(3

300

150

B. Cellular laminin after trypsin

C. Secreted laminin

200 -

100 -

I 100 150 200

FRACTION NUMBER FIG. 10. The oligosaccharide content of cellular and se-

creted laminin. JAR cells were labeled continuously with [3H] mannose for 20 h. Cellular laminin was immunoprecipitated from lysates of an equal number of either control cells or trypsin-treated cells; secreted laminin was immunoprecipitated from the labeling medium. The immunoprecipitates were digested with protease and then with endo H, and the array of labeled oligosaccharides was fractionated by gel filtration chromatography as a column of Bio-Gel P-4. Ovalbumin (oual) and mannose (man) were added to the samples to mark the void and included column volumes, respectively. The elution positions of the oligosaccharide standards MaQGlcNAc, Man7GlcNAc2, ManGGlcNAc, and Man5GlcNAc are indicated by the peaks numbered 8, 7, 6, and 5, respectively. The endo H-resistant glycopeptide fraction is designated as GP. A , cellular laminin from control cells; B, cellular laminin from trypsin-treated cells; C, secreted laminin from the labeling medium.


alteration of high mannose oligosaccharides to sialidase-sensitive complex oligosaccharides (Fig. 6, Miniprint Section).

A single A precursor molecule (PA) gives rise to a pair of mature A forms (A and A’), and a pair of B precursor molecules (pB1 and pBz) gives rise to a pair of mature B forms (B1 and Bz). The B doublet has been observed previously by others in many (23-25, 34, 36, 67) but not in all (1, 2, 4, 26- 28, 32, 33) laminin preparations, particularly when cultures of human (36) or murine (23-25, 34, 67) cells were biosynthetically labeled with radioactive amino acid precursors. The persistence of two B subunits in tunicamycin-treated JAR (Fig. 5, Miniprint Section) and murine embryonal carcinoma cells (25, 67) shows that the key difference between the B doublet forms is not due to N-linked oligosaccharide substituents. Indeed, two distinct laminin B subunit products have been observed upon in vitro translation of mRNA from murine parietal yolk sac carcinoma (PYS-2) cells (67), suggesting that the B doublet forms are distinct polypeptides coded by separate mRNA species. Both B subunit forms participate in the formation of the laminin molecule (Fig. 7).

The A subunit of laminin secreted into the culture medium migrates as a doublet (A and A’) on SDS gels, whereas the cell-associated mature laminin contains principally the A form, corresponding to the larger band of the A doublet. The appearance of A’, the smaller band of the A doublet, is closely timed with secretion and seems to be the result of limited proteolytic action on the A subunit, because A’ migrates even more rapidly on SDS gels than the precursor molecule (PA) from which it was formed. A proteolytic event might desta- bilize the binding of laminin to the cell surface or to other matrix elements, thereby accelerating its secretion into the culture medium. This suggests that the mature cell-associated and secreted JAR laminin forms may differ in the ability to promote the attachment of epithelial cells to their substrata. Based on the relative intensity of A to A‘ in secreted laminin, only about one-third of the secreted laminin molecules contain the modified A’ subunit. Proteolysis of A, therefore, does not seem to be obligatory for laminin secretion. It may be, however, that three or four cell surface laminin molecules are associated to form a supramolecular complex that is released by the modification of a single A subunit of one of the constituent laminin molecules.

Processing of the N-linked Oligosaccharides of Laminin: Pinpointing the Rate-limiting Step-Laminin resembles secretory (68) and integral membrane glycoproteins (68) in the overall pattern of N-linked oligosaccharide biosynthesis. The conversion of high mannose oligosaccharides to sialic acid- containing complex oligosaccharides distinguishes, at least in part, the precursor and the mature forms of the laminin subunits. The rate-limiting step in laminin processing by JAR cells is the trimming of the high mannose oligosaccharides from MangGlcNAc2 units to Man,GlcNAc2 units, catalyzed by the Golgi-localized form of a-mannosidase I (66). This was shown in continuous labeling experiments by the accumulation of intracellular laminin precursors containing mostly MangGlcNAcz and MansGlcNAc2 units (Fig. lo), and in pulse- chase kinetic experiments by the slow trimming of these o l igosacchar ides (F ig . 9 ) . The MangGlcNAcz and MansGlcNAcz units are the products of the processing enzymes glucosidase I, glucosidase 11, and mannosidase I (endoplasmic reticulum form), known to be localized in the rough endoplasmic reticulum (69-71). The accumulation of precursors bearing these oligosaccharides suggests that laminin forms reside in the rough endoplasmic reticulum during most of their intracellular life (72-76). Immunoelectron micro- scopic examination of murine cells that produce laminin has

shown prominent staining of the cisternae of the rough endoplasmic reticulum with anti-laminin serum (31, 77-80), consistent with the idea that laminin precursors accumulate in that organelle. Thus, the rate-limiting step in laminin processing appears to be translocation from the site of synthesis in the rough endoplasmic reticulum to the Golgi complex, the site of terminal oligosaccharide processing. Our previous studies have shown that the secretory glycoprotein hCG, also produced by JAR cells, encounters the same rate- determining step as laminin in the processing of its N-linked glycans (57, 58). The a and /3 subunits of the hCG a/3 dimer, like the A and B subunit precursors of laminin, accumulate in JAR cells as precursors bearing mostly MangGlcNAc2 and MansGlcNAcz oligosaccharides, consistent with the slow translocation of hCG forms from the rough endoplasmic reticulum to the Golgi complex. Once this slow step is traversed, processing to the N-linked glycans to complex chains is com- pleted rapidly (approximately 15 min) for both laminin and hCG, and the mature forms of both glycoproteins are continuously externalized by the cell without intracellular storage.

It is interesting to note that a-mannosidase I (presumably the endoplasmic reticulum form of this enzyme) acts on the different glycoprotein subunits of laminin and hCG at different rates and to different extents. For example, the oligosaccharides of the laminin B precursor and the hCG-a precursor are trimmed to MansGlcNAc2 forms and then to smaller forms, predominantly Man6GlcNAc2, prior to traversing the rate-limiting step, whereas the oligosaccharides of the laminin A precursor and the hCG-/3 precursor are not significantly trimmed beyond the MangGlcNAcz point. This could be due to the “substrate activity” of the different glycoprotein subunits, governed by the steric accessibility of the N-linked glycans to the rough endoplasmic reticulum form of mannosidase I. In addition, the laminin B subunit resides in the rough endoplasmic reticulum for a longer period of time than the A subunit (Fig. lA), giving the rough endoplasmic reticulum form of mannosidase I more time to act on this substrate.

Assembly of the Laminin Molecule-Since murine laminin subunits appear to be translated as separate polypeptides from distinct mRNA r,ecies (67), the combination of the subunits to form the laminin molecule presumably occurs as a post- translational event. Newly synthesized A subunit is rapidly incorporated into the fully assembled laminin molecule as shown 1) by the early appearance of laminin in cell lysates following a short biosynthetic pulse (Fig. l ) , and 2) by the association of most of the labeled A subunit with the M, -950,000 laminin molecule (Fig. 7). In contrast to A subunit, newly synthesized B subunit is recovered from cell lysates predominantly in the form of uncombined pB subunit or disulfide-linked pBlpB2 dimer (Fig. 7). This suggests that the capacity of JAR cells to produce laminin is limited by the availability of the pA subunit such that the pB subunit forms, produced in excess, accumulate intracellularly.

The structure of laminin, as indicated by rotary shadowing electron microscopy (7), seems to be an A B 3 tetramer with the A subunit comprising the long arm and three B subunits comprising the short arms of the cross-shaped molecule. We have observed in the pulse-chase experiments no laminin forms that correspond to B3 or AB2, the anticipated intermediates in the assembly of a tetrameric molecule, suggesting these forms are either immunologically unreactive‘ or are present in low concentration at steady state. Alternatively, Howe and Dietzschold (34) have suggested an AB2 trimer structure for the laminin molecule. Although our kinetic results do not rule out the AB8 tetramer model for laminin, they are consistent with and more easily explained in terms of the


CpBll + p B c " p B l p B z

[PA]"/ for pB,pB, t ranslocat ion of select ive pAPB,PB,

FIG. 12. Model for the biosynthesis and secretion of laminin by JAR choriocarcinoma cells. - PAPBTPBZ -

endoplasmic reticulum

limited

proteolysis

N-linked (A-A'), maturation of

ol igosaccharides secretion

ABlBz

A'B,B, CABIBzI e ABlB2 - i- u u U

Golgi cell culture complex surface medium

[ 1. intermediates in low concentrat ion

e, t ranslocal ion between compartments, secret ion

@I, rate determining s tep f o r processing and secret ion

AB2 trimer model, as depicted in Fig. 12. Intracellular Translocation, Externalization, and Secretion

of Laminin-The assembly of the disulfide-bonded laminin molecule, like the noncovalent hCG a@ dimer (58), occurs in the rough endoplasmic reticulum while the respective subunits contain Man9GlcNAc2 and MansGlcNAc2 oligosaccharides. A key difference between laminin and hCG, however, is that free, uncombined hCG subunits ( a and @) are processed and secreted along with the hCG a@ dimer (58), whereas the uncombined laminin pB and pB1pB2 dimer forms are neither processed nor secreted to any measurable extent. For laminin, but not for hCG, subunit assembly seems to be a prerequisite for translocation out of the rough endoplasmic reticulum. In this regard, laminin resembles other multisubunit glycoproteins such as HLA antigens (81-83), macrophage cell surface glycoprotein (Mac-1) (84), or immunoglobulin M (85), molecules that must be assembled prior to maturation and/or externalization by the cell. The differential processing and secretion of laminin compared to pB dimer or uncombined pBlpB2 are inconsistent with the bulk translocation of the lumenal contents of the rough endoplasmic reticulum and suggest a mechanism, perhaps receptor-mediated, for the se- lective transfer of the assembled molecule from the rough endoplasmic reticulum to the Golgi complex. It is conceivable' that this event is mediated by molecules such as the laminin receptor or sulfated glycolipids, integral membrane molecules with which laminin can specifically interact (13-18). Recep- tor-mediated intracellular translocation events have been pos- tulated to explain observations that different glycoproteins are secreted with different kinetics by the same cell (86,-87) as are hCG and laminin by JAR cells (58);

The apparent paradox that A subunit matures more rapidly than the B subunit forms in the pulse-chase experiment in Fig. lA can be explained by the observations that 1) B subunit forms are produced in excess over A subunit forms and 2) uncombined B subunit forms (pB and pBlpB2 dimer) that accumulate intracellularly (Figs. 1B and 7) are less efficiently translocated to the Golgi complex than the assembled laminin molecule (see Fig. 12). Newly synthesized PA, labeled with [35S]methionine during a 10-min pulse, rapidly combines with unlabeled pBlpB2 from the pre-existing intracellular pool to form laminin which is translocated to the Golgi and processed to maturity with a half-time of 1 h. In contrast, newly synthesized pB is sequestered intracellularly as pBlpB2 dimer (Fig. 7). Since pBlpB2 is inefficiently translocated to the Golgi complex, its further processing is limited by the availability of pA with which to combine. Therefore, the pBlpB2 is processed more slowly than PA, with a half-time of 4 h.

A major difference between hCG and laminin is that bio-

synthetically labeled hCG is totally secreted into the culture medium by 4 h of chase (58), whereas 71-77% of labeled laminin remains associated with the cells at 8 h of chase. Most of the mature laminin externalized by JAR cells remains bound to the cell surface, perhaps via the laminin receptor or deposited in an extracellular matrix, and is only slowly secreted into the culture medium, a process that may be aided by limited proteolysis of the A subunit. This dramatic difference in distribution of the two externalized glycoproteins between the cell layer and the culture medium could be interpreted in terms of their biological functions: laminin appears to be a prominent cell surface component which may function in cell adhesion, whereas hCG, as a hormone, is transported in the blood to its target tissue.

We are currently attempting similar biosynthetic studies with explants of human placental tissue in order to ascertain potential differences in the biosynthesis and matrix deposition of laminin by malignant and nonmalignant human troph- oblast cells.

Acknowledgments-Randy Knibbs prepared the mouse EHS tumor laminin in our laboratory (I. J. G.). Linda Harbison skillfully typed this manuscript.

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Laminin Synthesis by Human Choriocarcinoma Cells 14741 SUpplenenral Information

to

The Biosynthesis, Processing, and Secretion of lamlnln by Hman Choriocarcinoma Cells

by

Barry P. Peters, Richard J. Hartle, Raymond F. Krzesicki, Todd G. Kroll Fulvio PermL,James E. Balun, I m l n J. Goldstein, and Raymond W. Ruddon

EXPERIMENTAL PROCEDURES

tunicmycin (National ~ r c d u c f s Branch, ~ational Cancer Institute1 at a f m a l concentration of The effect of tunicalnycin On the laminln Subunits. JAR cultures Yere treated with

5 g/ml In complete growth medium for 16 h prlor to metabolic labeling of the cells wlth I3'Simethionine. We have shown that this treatment blocks the E-glycosylatian of hCG in JAR cells while rnhibiring protein Synthesis by 20 percent 1551. Tunicmycin was added to the cultures 8s a concentrated solution I1 nq/mll in DMSO; an ewal volume of DMSO was added eo conrro1 cultures.

Digestion of lminin with sialidase and endo H . The washed Protein A-Sepharose imunoprecipitates containing the adsorbed laminin farms were 5pllt info two equal portions and resuspended ~n the appropriate buffer., The glycosidase was added to one of the duplicate samples, while the other served as a control. The Protein A-sepharose suspensions were incubated wlth aqiratlon at 37'C for 16 h. The treated lnmunoprecipitares were then

manner. Sensitivity Of the laminin forms fO the glycosidase was ]udqed by a shift in collected by centrifugation, washed twice in P85 and analyzed by BDS-PAGE I" the usual

mobility on SDS-PAGE compared to the control lacking enzyme. The digestions with endo H IMlleS) were carried Out with 10 mU Of enzyme in 0 . 5 ml Of 0.1M sodium citrate, pH 5.3,

phenylmethane~ulfonylfluoride 12 mM1 In Order to inactive residual protease actlvlty. The containing 0 . 0 4 percent scdim azide. The end0 H preparation had been pre-treated with

digestions with sialidase ISiqma, Type "111 from Clostrldim rfrinqensl were carried out wlth 50 nU of enzyme In 0.5 ml of 0.05" s c d i u m ~ H ~ . O , containing 0.04 percent sodium azide.

RESULTS

sarcoma IEHSI. The imunoreactive l,yninln form5 were precipitated from JAR cell lysates A comparison of laminins isolated from human Choriocdrclnona (JAR1 cells and mvrine

after a 1-h bioIynthetic PYlse wlth I Slnethionine. f r o m cell lysates after a 4-h Chase with non-radioactive growth medium, and from the 4-h chase medlm. The nobilirles of J l i R laminin and the murlne EH tumDr laninln On SDS-PAGE Were compared IF lg . 2). Under nan-reducing conditions. the 155Slmethionine-labeled JAR lanunin secreted I n t o the 4-h chase medlm co-migrated wirh the EHS laminin rnolecule IM 9. 950,0001 (Fig. 2, lane 1 Y S 2 1 . A f t e r

to the characteristic A (Mc Z 400,OODl and B IM = 200,0001 Subunit types IFlg. 2, lane 5 YS

reduction of disulfide bonds with 2-nercaptoeth5nol. the $ m e JaR imunoprecipitate gave rise

61.

Ent - . . Ent -

1 2 3 4 5 6 - \ Y 2

non-reduced reduced CM PC CC CM

JAR E H S JAR E H S -

F i g . 2. Imunoreactive la~inin forms from hman choriocarcinoma cells. Two 100 m djghes of JAR ce l l s 12 x 10 cells per dish1 were biosynthetically labeled for 1 h with

for an additional 4-h incubation. The lysates from the I-h pulse cells (PC) and 4-h I Slmethionlne, and one of the dishes was chased with non-radloactlve culture medim

chase cells ICCI. and the 4-h chase medium ICMI were imunoprecipitated with

percent polyacrylamrde gradient slab gel. The rmunoreactive lminin farms from JAR anti-laminm serum, and the imunoprecipitates were analyzed by SDS-PhGE on a 3-10

cells were compared to the laminin lsolared from the mouse EHS tumor before ( lanes 1 and 21 and after (lanes 3-61 reduction Of disulfide bonds vlrh 2-nercaptaethanol. The

moUSe lminln was detected On the gel wlth the sxlver stain. Lane 1 , 4-h chase nedim: radiolabeled bands of human laminin were vlsualired by fluorography of the gel, and the

lane 2 , EHS tumor laminln; lane 3 , 1-h pulse cell lysate: lane 4 , 4-h chase cell lysate;

molecule; A, A', 0 and B represent mature lamlnin subunits. pa pn and pB lane 5 . 4-h chase medrum; lane 6 , EHS tumor l m i n l n . lam indicates the laminin

represent the lamin!, subvniz prec~rsor forms: Ent Tepzesents tie ba'semed; membrana g1ycoprorern entactin.

"on-reduced ENS laminin (Fig. 2 , lane 21 conrained in addition to the intact lminin molecule The secreted JAR lminin and the EHS laminin differed in several respects (Fig. 21: the

a M 2 400 000 form that may be uncombined A Subunit Or a disulfide-linked B dimer. whereas JRRrlamlniA did not (Fig . 2 , lane 11. The reduced A and 8 subunits O f JliR laninln migrated as doublets on SOS-PIIGE whereas the EHS subunlts mlgrated as broad single bands (Fig. 2 , lane 5 VI 61. The reduced JRR laminin A doublet migrated slightly more rapidly on SDS-PAGE than the reduced EMS l a m i n A subunlt (Fig. 2 , lane 5 vs 61. In addition, the basement membrane glycoprotein entactin, present in the EHS l m i n r n preparation, was absent in the JAR lminln im"noprecipirate*.

The specificity of lminin imvnoprecipifation. Two controls were employed to verify thar the rabbit anti-EHS lmlnin antiserum Ipecrfically precipitated mmunoreactlve l m l n l n polypeptides from JAR culture flulds. First, when nom--"ne serum vas substituted for ant i - laminm serum, no radioactive polypeptides were seen on the SDS ge l fluorograph IFiq. 3, lane 3 VI lane 1 ) . second, an e x c p p of Ens laminin added to the detergent lysates blocked

addition. Flqure 3 illustrates that the JAR culture~ were not producmg fkbronectin, a the lmunopreElpitatian Of the I Slmethionine-labeled JAR laminin forms Wig. 41. In

possible contaminant in EHS laminin preparations. &nti-flbronecrln antiserm did not preclpitate labeled polypeptidelsl from JAR cell lysate (lane 21. Fibronectin-praducing human melanoma and rhabdomyosarcoma cells, tested eo verify the activity of the antl-fibronectln antiserum, synrheslzed lmunoreactive polypeptides that mlgrated with the characteristic M of fibmnectin both With and without reduction of disulfide bonds.

A. NON-REDUCED

-" 1 2 3 4 5 6 7 8 9

B. REDUCED

"- 1 2 3 4 5 6 7 8 9

JAR AW35 A673 Fig. 3. Human choriocarcinoma cells do not synthe9ize frbronectin. Cultures of JAR cells, AW735 human melanoma cells. e d A673 hman rhabdomyosarcoma cells in 100 m dishes were labeled for 30 nin with I Slnethionine. The detergent lysates Of each cell cvlture were drvrded lnto three equal pr t ions . imunoprecipltated vrth anti-laminm (lanes 1,4,1), antL-fibronectin (lanes 2 . 5 . 8 ) . or "on-mmune serum (lanes 3.6.91 and analyzed by SDS-PAGE without (panel A) or with (panel 81 reduction with 2-mercaptoethanol. The precursor forms of the lminin A subunit (PA), the lanlnin B EUbUDltJ IPS and pS ) and fibronectin (Fn) are indicated by the arrowheads: lpB12 de*iqnates th! disulf?dd-linked dllner form of PB..

Lam -


N-qlycosylation of prote~ns by inhibiting the flrst step in the dolichol pathway, the The effect of tunicamycin on lmin in biosynthesis and secretion. Tunicarnycin blocks the

synthesis Of N-~cerylqlucosaminylpyrDphosphoryl dolichol (88,891. We have previously shown that JAR cell; treated with rvnicalnycin I 5 vq/nll synthesize the a and B Subunits of the qlycOprotein hornone chorionic gonadotropin that lack E-lmked carbohydrate chains, and consequently rnlgrate more rapldly on SDS-PAGE than the fully qlycosylated Subunits (551. The bio5ynrhesis of laminin in J A R cells was similarly affected by tunicanycm. Both the A and B subunits f r m tunicanycin-rreated culture3 algrated more rapidly on SOS-PAGE than their Counterparts from control cultures (Fig. 5, lane 5 YS 1. lane 13 vs 9, lane 21 vs 171, consistent with the presence of x-linked glycans in both subunits. The incorporation of

l'5Slmethionine into the laminin polypeptides was Similar in the conr1.31 and tunicamycin- Created cultures (Fig. 5 , lane 1 vs 5). The secretion of laminin into rhe chase medium was inhibited In the tunicamycin-treated cells (Fig. 5 , lane 21 vs 171. HCG secretion by JAR cells is not slgnif>canfly inhibited by 5 Yg/ml tunicamycin (551. longer exposures Of the fluorograph in panel C revealed that both the cellular and secreted laminin forms from the tunicamycin-treated C Y ~ C Y I ~ S had the same apparent mobility on SDS-PAGE (data not shown).

-

A. Pulse cells 6. Chase cells C. Chase medium

- 200K

- 1 2 3 4 5 6 7 8 910111213141516

- 17 18 1920 2 122

-91K

- TU + T U - T U + T U - TU + T U

Fig. 5 . The effect of tunicmycin and endo H on the innunoreactive laminin forms produced by human Choriocarcinma cells. Two 100 m dishes of JAR cells were treated with tvnicalnycin at a concentration Of 5 yq/ml (+TU), and two dishes were treated vrth the dilpgibylsulfoxide vehicle alone (-TU) for 16 h prior to labeling the cells for 1 h with [ Slnethionine. One dish of each group was then chased with non-radioactwe culture medium for an additional 4-h incubation. The I-h pulse cell lysates, 4-h chase cell lysates and 4-h Chase media were divided into three equal aliquots. One aliquot was imwopreoipitated with anti-lammin serum (LAM: lanes 1, 5, 9, 13, 17 end 21); another aliquot -as imunoprecipltared with non-imune IabbLt serum as a control (NRS: lanes 2, 6, 10. 14, 18 and 221. The anti-laminin imunopreclpitates from the third aliquot were resuspended in sodium citrate buffer, divided into two equal

Y S 8, 11 vs 12, 15 YS 16 and 19 vs 201. A, A , , B1, ,and 0% ivdicate the mature laminin

portions, and incubated for 16 h without or with 10 mU of endo H (*EH: lanes I VS 4 , 7

forms, while PA, pB and pB designate the lanlnln 4u Unlt PrecYI'sOr forms. The imunopreclpitares d e reduced with 2-mercaptoethenol and were analyzed by SOS-PAGE on a 5 percent polyacrylamide slab gel. The arrows show the aiqratlon Of the mOleCYlaT weight markers myosin (M = 200 0001 and phosphorylase IK = 91 000).

Panel A , 1-h pulse $ells; panel B, 4-h Chase cells; 6an.1 c, 4-h Chase media. The effect of end0 H on the laninin subunits. The glycosidase endo H cleaves the

chitobxosyl core Of high annose but not Of complex N-linked oligosa~charides (90,911. Endo H treatment Caused the 13~Slmethi~nine-1~beled cel luiar A and B plecursorI (PA PB and PB 1

of the secreted mature laninin Subunits IFiq. 5 , lane 19 vs 201. Therefore, endo H-sensitive eo migrate more rapidly on SDS-PAGE (Fig. 5 , lane 3 vs 41 but had no effect 0. th! nobiligy

hzqh mannose Oli40saCChaltides are Substituents of cellular A and B precursors but are either absent from or cryptic to endo H I" the intact mature subunits. The lminin precursors deglycosylated by endo H migrated similarly, but not exactly. on SDS-PAGE eo the respective laminin f o m s produced by tunicanycin-treated cells (Flg. 5, lane 4 VI 51 . The lanininm forms from tunicanycin-treated cells were mostly unaffected by endo H IF1g. 5, lanes 7 vs 8 , lane 15 vs 16) . However, the lass of a portion of the A subunit (Fig. 5, lane 7 vs 81 nay be due

nethanesulfanylflvoIide pre-treatment. to residual proreolytlc activity ~n the endo H preparaeion even after the phenyl

chase cell lysates allowed a direct comparison of their mbilities on the same SDS-gel lane. A mixture of precur+or and mture laminin forms in the immunoprecipitate of the 4-h

The doublet Of mature B nlgrated more SlWly than the doublet of the PIeCurIOI B subunit, although the smaller of the mature R forms overlaped the larger of the precursor B forms

endo H. Under these conditions, the precursor doublet migrated mom rapidly, and was (Fig. 5, lane 111. ThlI 1s more clearly seen after the Imunoprecipitate was digested with

resolved f r m the unperturbed mature B chain doublet (Fig. 5 , lanes 11 and 121. The precursor form of the R chain niqrared between the two bands of the mature A doublet (lane 91.

terminal slallc acid groups that are camon substituents of complex :-linked oligosac~haride~ The effect of sialrdase on the laminin Subunits. Sialidase cleaves the nonreducing

but are absent from hiq3pannose Oligosaccharide Chalns. Sialidase treatment had no effect on the mobility of the I Slmethionine-labeled cellular A and B precursors (PA, pB and pB ) (Fig. 6, lane 1 Y S 2). bnt; caused the secreted mature lanmm forms R A ' B land B $0

migrate more rapidly on SOS-PAGE (Fig. 6, lane 5 vs 61. Therefore, rh.'.i,iid);.-..".itive complex oligosaccharides are present in the secreted mature A and B laminin Subunits. but are absent lor cryptic to sialidarel in the cellular PT~CYTSOT forms of laminin.

1 2 3 4 5 6 Sial idase: - i- - i- - i- "-

Pulse Chase Chase cells cells medium

Fig. 6. The effect of sialidsse on the irmnunoreacrive laminin forms proqyced by human Choriocarcinoma cells. W o 100 mm dishes of JW? cells were labeled with 1 Slnethionine

additional 4 h of incubation. The anti-laminin ~munoprecipirates from the 1-h pulse for 1 h, and one of the dishes was chased with nan-radioactive culture medim for an

divided into twC, equal portions, and incubated for 16 h without Or with 50 mu Of cells, 4-h chase cells and 4-h chase medium were resuspended in sodium acetate buffer,

Clostridium perfrrnqens slalidase. The treated imunoprecipltates were reduced with 2-merceptoethanol and analyzed by SDS-PAGE on a 3-10 percent acrylmide qradient slab gel. A , A ' , B designate the lamlnln SY u n l r precurso~ forms.

,and 8% indicate the mature laminin farms, while PA, pR1, and PB2

asSOClated wlth the JAR cell layer was labeled Y L t h anti-laminln antiserm followed by The deposition of laminin in a basal lmina-like matrix. The extracellular laminin

fiuorescem-conjugarea second antibody and v~suallzed by fluorescence ~icroscopy. The protocol Yap carried out with viable cells at 4'C in order LO m~ninize the intracellular a C C m Y l a t i O n Of fluorescent anflbody and t o accentuate the labeling of extracellular structures. The anti-laminin serum stained punctate, patchy clusters of e X t T I C e l l U 1 I I r naferral (Pig. 11R) rhet were not stained by non-imune serum (Fig. 118).

OF CHEMISTRY No. of November 25, pp. 1985 by Inc U.S.A ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0...

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Transcript of OF CHEMISTRY No. of November 25, pp. 1985 by Inc U.S.A ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0...