©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
COOH-terminal Proteolytic Processing of Secreted and Membrane Forms of the Subunit of the Metalloprotease Meprin A
REQUIREMENT OF THE I DOMAIN FOR PROCESSING IN THE ENDOPLASMIC RETICULUM (*)

(Received for publication, October 27, 1994)

Petra Marchand Jie Tang Gary D. Johnson Judith S. Bond (§)

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the ``I'' domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alphabeta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.


INTRODUCTION

Meprins are plasma membrane metalloendopeptidases of the ``astacin family'' that are expressed at high concentrations in brush-border membranes of mouse and rat kidney and of mouse, rat, and human intestine(1, 2, 3, 4, 5, 6, 7) . They are glycosylated homo- or heterotetrameric proteins of alpha and/or beta subunits(8) . The mouse alpha and beta subunits are evolutionarily related (approximately 42% overall sequence identity), and their cDNAdeduced primary sequences predict a similar arrangement of functional domains.

Both transcripts encode a transient NH(2)-terminal signal sequence (S) and a prosequence (Pro) preceding the catalytic protease domain. Following the protease domain is a MAM domain, proposed to be an adhesion domain which has been identified as a component of several cell surface proteins including meprin, A5 protein, protein tyrosine phosphatase µ, and enteropeptidase(9, 10) . Near the COOH termini, both nascent proteins have an epidermal growth factor (EGF)(^1)-like, a hydrophobic putative transmembrane-spanning (T), and a cytoplasmic (C) domain. Between the MAM and EGF-like domain is a large domain (182 amino acids for alpha, 176 amino acids for beta) of unknown function, the X domain, which has no known homology to other proteins. The two transcripts differ in that alpha encodes a 56-amino acid sequence between the X and EGF-like domains that has no counterpart in beta, indicated above as the inserted (I) domain(11) .

Previous results indicate that the biosynthetic maturation of meprin subunits involves NH(2)- and COOH-terminal proteolytic cleavages and that the mouse kidney alpha and beta subunits are differentially processed in vivo(11) . During biosynthesis of the alpha subunit in the mouse kidney, the signal sequence and the prosequence are removed from the NH(2) terminus, whereas the mature beta subunit retains the prosequence and thus has latent proteolytic activity. Differences in COOH-terminal processing determine the expression of membrane-bound and secreted forms. The mature beta subunit retains all COOH-terminal domains and is an integral type I membrane protein. The mature alpha subunit contains neither the COOH-terminal membrane anchor (T) domain nor the EGF-like domain predicted from the alpha subunit transcript; these domains are removed by co- or posttranslational proteolytic cleavage(s). Thus, beta subunits are always membrane-bound, whereas alpha subunits are either membrane-associated (if complexed with beta subunits by disulfide-linkages or adhesion) or secreted into the urine (as homooligomers). This type of oligomeric organization is unique among cell surface proteinases.

The enzymes responsible for NH(2)- and COOH-terminal proteolytic processing and the subcellular sites of processing are not yet known. However, there is evidence that cultured cells could be useful for investigating COOH-terminal processing events. Expression of rat and human meprin alpha subunit transcripts in human 293 cells, COS-1 cells, or Madin-Darby canine kidney cells leads to secretion of the subunit into the culture medium as a zymogen(12, 13, 14, 15, 16) . These and other data indicate that the alpha subunit is processed at the COOH terminus in cultured cells as in vivo but that the NH(2)-terminal prosequence is not removed in cultured cells as in vivo, and thus these cells produce an inactive meprin alpha subunit. The amino acid sequence of the mouse alpha subunit is 86% identical to the rat subunit and 75% identical to the human sequence, and the expectation is that mouse subunits are processed as the rat and human counterparts.

Molecular weight estimates of deglycosylated meprin subunits from mouse kidney indicated that the processing of alpha occurs within or very close to the I domain(11) . Furthermore, this domain contains two sites, a furin-site and a dibasic amino acid site, that are potential candidates for processing(17) .

In order to determine the role of the alpha subunit I domain in processing, chimeric and mutant meprin alpha subunits were expressed in COS-1 and human 293 cells. In addition, experiments were designed to determine the subcellular site of COOH-terminal processing. The studies establish that processing of alpha subunits occurs early during biosynthesis in a preGolgi compartment and that the I domain is required and sufficient for COOH-terminal proteolytic processing.


EXPERIMENTAL PROCEDURES

Plasmid Constructions

cDNAs encoding wild-type or mutant mouse meprin subunits were cloned into the expression vector pcDNAI/Amp (Invitrogen). The wild-type rat meprin beta subunit and the rat meprin alpha c-myc construct were present in the expression plasmid pCMV1(18, 13) . Transcription from either plasmid is directed by the promoter of the human cytomegalovirus early gene. Full-length meprin alpha subunit cDNA was generated from two previously isolated overlapping clones, clone 723S (containing nucleotides 1-691) and clone B24 (containing nucleotides 204-2466), by polymerase chain reaction (PCR) amplification of the 5` end (nucleotides 1-655) using clone 723S as template, and ligation of the NdeI digested PCR product to the NdeI digested B24 insert(2) . The sense primer used for the PCR contained the consensus sequence for efficient eukaryotic translation initiation ((C/A)XXATGG; start codon, underlined) surrounding the start codon to maximize the level of expression(19) .

The amino acid substitutions in the I domain of the mouse alpha subunit and the deletions of the I domain or portions of the I domain were generated by site-directed mutagenesis following the method of Deng and Nickoloff (20) using the Transformer site-directed mutagenesis kit (Clontech). The mutagenic primers for the base substitutions were designed to change amino acids Arg/Lys to Gly/Gly (construct alpha645GG) and/or Lys/Arg to Gly/Gly (constructs alpha666GG and alpha645/666GG). The mutagenic primers for the deletion mutants contained the flanking regions surrounding a deletion of nucleotides 1897-2064 (construct alphaDelta628-683, deletion of nucleotides 1996-2064 (construct alphaDelta661-683), or deletion of nucleotides 1897-1995 (construct alphaDelta628-660). All clones were further analyzed for the desired mutations by DNA sequencing or restriction mapping. The mutations are shown diagrammatically (see Fig. 2Fig. 3Fig. 4).


Figure 2: Cell-associated expression of meprin alpha subunit mutants lacking the inserted domain. Schematic diagrams show wild-type (wt) and mutant meprin subunits that were transfected into COS-1 cells. The individual domains are indicated by the abbreviations: P, protease domain; MAM, adhesion domain; X, unknown domain; I, inserted domain; E, epidermal growth factor-like domain; T, transmembrane domain. The alpha subunit domains are shown in white; shadedareas represent mouse beta subunit sequence. All samples were subjected to SDS-PAGE in the presence of beta-mercaptoethanol, transferred to nitrocellulose, and probed with an anti-meprin alpha subunit antibody. PanelA, Cellular membranes (cells) and media from cells transfected with either wild-type meprin alpha subunit cDNA or mutant cDNAs; panelB, cellular membrane fractions before and after treatment with Endo F or Endo H; panelC, membrane fractions isolated from transfected COS-1 cells were treated with sodium carbonate, and supernatant and precipitated fractions were prepared as described under ``Experimental Procedures.''




Figure 3: Expression of mutant rat meprin beta subunits with inserted mouse alpha subunit I domain or substituted COOH-terminal domains. Schematic diagrams show the wild-type (wt) and mutant meprin subunits that were transfected into COS-1 cells. Rat beta subunit domains are shown in gray; mouse alpha subunit sequences are shown in white. Tissue culture media and cellular membrane fractions from COS-1 cells transfected with the wild-type or mutant beta subunits were subjected to SDS-PAGE in the presence of beta-mercaptoethanol. Meprin subunits were detected with anti-rat meprin antibodies.




Figure 4: Expression of mutant meprin alpha subunits containing amino acid substitutions or deletions within the inserted domain. Top, schematic diagrams of the wild-type and mutant meprin subunits. The wild-type mouse alpha subunit contains two dibasic sites within the inserted domain at positions Arg/Lys and Lys/Arg. In the substitution mutants, one or both dibasic sites were replaced with double glycine residues, as indicated. The deletion mutants lack either the amino-terminal 33 amino acids of the I domain (residues 628-660) or the COOH-terminal 23 amino acids of the I domain (amino acids 661-683), as indicated by the horizontal bars. Samples from cells transfected with wild-type or mutated meprin alpha subunit cDNA were subjected to SDS-PAGE in the presence of beta-mercaptoethanol; immunoblots were analyzed using antimeprin alpha subunit antibodies.



COOH-terminal deletion mutants for the meprin alpha subunit were generated by PCR using mutagenic antisense primers, which changed the codons for Leu (construct alphaDelta26), Arg (construct alphaDelta76), or Arg (construct alphaDelta133) to stop codons. A diagram for the mutations is shown (see Fig. 5).


Figure 5: Expression of mutant meprin alpha subunits lacking portions of the COOH terminus. Top, schematic diagrams of the wild-type and mutant meprin subunits. Bottom, concentrated tissue culture media and membrane-enriched fractions from cells transfected with either vector DNA (control), wild-type meprin alpha subunit cDNA, or truncated transcripts were subjected to SDS-PAGE in the presence or absence of beta-mercaptoethanol, followed by immunoblot analysis using anti-meprin alpha subunit antibodies.



To generate the meprin alpha/beta subunit hybrid cDNA encoding mouse alpha subunit residues Met^1-Ser fused in-frame to mouse beta residues Pro-Phe, a TthIII site present at position 1832 of the mouse beta cDNA was used (see Fig. 2). Full-length mouse meprin beta subunit cDNA, obtained by reverse transcriptase PCR from C3H/HeJ mouse kidney RNA, was cloned into pcDNAI/Amp and digested with TthIII and EcoRI to excise nucleotides 1-1832. A TthIII site was introduced into position 1880 of the meprin alpha subunit cDNA by PCR; the PCR product was digested with TthIII and EcoRI and subsequently ligated into the TthIII-digested pcDNAI/Amp vector containing the beta subunit 3` end.

The chimeric constructs beta/alpha and beta/alpha/beta were generated by recombinant PCR with primers containing overlapping ``5` add on'' sequences according to the method of Higuchi (21) using mouse alpha and rat beta cDNA. The beta/alpha construct encodes rat beta subunit amino acids Ala^1-Thr fused in frame to the mouse alpha subunit residues Arg-Gln. The beta/alpha/beta construct encodes rat beta subunit residues Met^1-Thr, mouse alpha subunit residues Arg-Tyr, and rat beta subunit residues Val-Phe fused in-frame. A diagram of the constructs is shown in Fig. 3. All PCR reactions were performed with Pfu DNA polymerase (Stratagene) or Ultma DNA polymerase (Perkin Elmer) to minimize the possibility of base misincorporations. The nucleotide numbers and amino acid numbers refer to the published sequences for mouse alpha, mouse beta, and rat beta subunits(2, 3, 4) .

Tissue Culture and Transfection

COS-1 cells (ATCC 1650 CRL) and 293 cells, an adenovirus-transformed human kidney cell line (ATCC 1573 CRL), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin (complete DMEM) in a 37 °C incubator containing 5% CO(2). COS-1 cells were transiently transfected using the DEAE-dextran method(22) . Briefly, 24 h before the transfection, logarithmically growing COS-1 cells were plated in 100-mm tissue culture dishes at 5 times 10^6 cells/dish. The next day, the cells were incubated for 30 min at 37 °C with 2 ml of transfection mixture (containing 10 µg of plasmid DNA and 500 µg/ml DEAE-dextran in phosphate-buffered saline, pH 7.4). The mixture was then removed from the cells, the cells were grown for 2.5 h in complete DMEM, supplemented with 80 µM chloroquine, and treated for 2 min with 10% dimethyl sulfoxide in complete DMEM. The cells were then allowed to grow in complete DMEM. Thirty-two h after transfection, the medium was replaced with serum-free medium; DMEM supplemented with insulin (2.5 µg/ml), transferrin (17.5 µg/ml), 20 mM ethanolamine, bovine serum albumin (1 µg/ml), soybean trypsin inhibitor (10 µg/ml), and aprotinin (10 µg/ml). Forty-eight h after transfection, the tissue culture medium was harvested, centrifuged for 20 min at 16,000 times g to remove cell debris, and concentrated 25-fold using Centriprep-30 and Microcon-30 concentrators (Amicon). The transfected cells were harvested by scraping in 2 ml of ice-cold phosphate-buffered saline and lysed by homogenizing in a hypotonic buffer (10 mM Tris-HCl, pH 7.5, containing 0.1 mM iodoacetamide, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 10 mM EDTA) at 4 °C (23) before they were homogenized with a Teflon glass homogenizer. After homogenization, the salt concentration was increased to isotonic levels by the addition of 250 mM Tris-HCl buffer, pH 7.5, containing 0.25 M NaCl. Cell homogenates were centrifuged at 4 °C for 30 min at 100,000 times g, and the precipitant fractions (membrane-enriched) were dissolved in a solution of 0.2% SDS in 20 mM Tris-HCl buffer, pH 7.4. Soluble cell fractions were generally discarded after it was established that no immunoreactivity was detected in these fractions. Typically, one-tenth of the medium or membrane-enriched sample obtained from one 100-mm plate was added to each lane for SDS-PAGE.

Human 293 cells were transfected with plasmids by the calcium phosphate precipitation procedure(24) . Briefly, 30% confluent cells were cotransfected with 10 µg of expression plasmid and 1 µg of PVA1, a helper plasmid, which enhances expression(25) . The cells were incubated in complete DMEM and grown to 90% confluence prior to exposure to radiolabeled methionine.

Pulse-Chase Radiolabeling and alpha Subunit Immunoprecipitation

For pulse-chase labeling, following transfection, 90% confluent 293 cells were incubated for 40 min in methionine-free DMEM containing 50 units/ml of penicillin, 50 µg/ml streptomycin, and 4% fetal bovine serum. Cells were then incubated with 0.2 mCi/ml [S]methionine in the methionine-free medium for 10 min. The cells were washed with phosphate-buffered saline and incubated in Opti-MEM (Life Technologies, Inc.), which contains 5 mM methionine, for various times. At each time point, medium and cells were harvested. The medium was concentrated to 500 µl with Centricon-30 concentrators. The cells were lysed in 40 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, 2 mM EDTA, 0.2% Nonidet P-40, 1% Triton X-100, 0.2% SDS, 0.2% sodium deoxycholate, 1 mM PMSF, 100 µM 3,4-dichloroisocoumarin, and 10 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). Cell lysates were passed through a 25-gauge needle several times to disrupt subcellular particles and complexes and centrifuged in a microcentrifuge. Supernatant fractions were collected for immunoprecipitation.

To immunoprecipitate the radiolabeled alpha subunits, cell lysates were mixed with equal volumes of a solution containing protease inhibitors (1 mM PMSF, 1 mM EDTA, 100 µM 3,4-dichloroisocoumarin, 10 µM E-64) and incubated with 4 µl of anti-alpha antiserum or 0.8 µg of c-myc antibody (Oncogene Science) at 25 °C for 1 h. Concentrated media were mixed with equal volumes of lysis buffer and then treated as cell lysates. Then, 30 µl of protein A-Sepharose (1:1 suspension) was added to the mixture. After a 1-h incubation, the protein-A Sepharose complex was precipitated by centrifugation and washed twice with 1 ml of lysis buffer (at half the concentration given above), and once with 20 mM Tris-HCl, pH 7.5, containing 137 mM NaCl, 0.1% Tween-20. Finally, the immunoprecipitates were prepared for SDS-PAGE by suspending and boiling in 50 µl of SDS sample buffer. Generally, 25% of the sample obtained from one 100-mm plate was applied to each lane for SDS-PAGE.

SDS-PAGE and Immunoblotting

Proteins were subjected to SDS-PAGE in 9% polyacrylamide gels according to the method of Laemmli and Favre (26) and transferred to nitrocellulose membranes. Membranes were blocked overnight at 4 °C with 10% dry milk protein in Tris-HCl-buffered saline containing 0.1% Triton X-100 and probed with antisera raised in rabbits against deglycosylated mouse alpha subunits, referred to as ``anti-meprin alpha subunit antibodies''(11) , or to rat meprin, referred to as ``anti-rat meprin antibodies''(13) . The primary and secondary antibodies were added to the membranes for 1 h each at 25 °C in 5% milk protein, 1% bovine serum albumin in the same buffer. The secondary antibody, horseradish peroxidase-linked anti-rabbit immunoglobulin from donkey, was detected using enhanced chemiluminescence detection reagents (Amersham Corp.). Molecular weight markers used were alpha(2)-macroglobulin, beta-galactosidase, fructose-6-phosphate kinase, pyruvate kinase, and fumarase (Sigma).

Endoglycosidase Digestion and Sodium Carbonate Treatment

Cell extracts, concentrated media, or immunoprecipitated meprin subunits were enzymatically deglycosylated using either a mixture of endoglycosidase F and N-glycosidase F (Endo F) or endoglycosidase H (Endo H). The samples were denatured in a solution of 0.5% SDS, 0.1% beta-mercaptoethanol, and 1 mM PMSF in 50 mM Tris-HCl, pH 6.8, by boiling for 5 min. The Endo F digestion was performed in phosphate-buffered saline, pH 7.4, containing 0.25 mM PMSF, 10 mM EDTA, 0.1% SDS, and 0.5% n-octyl-beta-D-glucopyranoside, with 1.25 units of Endo F (Boehringer Mannheim). Endo H digestion was in 20 mM NaOAc buffer, pH 5.2, containing 0.1% SDS, 10 mM EDTA, and 0.25 mM PMSF, with 25 milliunits of Endo H (Boehringer Mannheim). Incubations for deglycosylation reactions were for 3-8 h at 37 °C; reactions were stopped by boiling the samples in SDS-PAGE sample buffer. Membrane-enriched fractions were treated with sodium carbonate as described by Fujiki et al.(1982). Briefly, membranes were precipitated by centrifugation for 30 min at 4 °C at 100,000 times g, suspended in 100 mM sodium carbonate, pH 11.5, and incubated for 30 min at 4 °C. The mixture was then centrifuged for 1 h at 4 °C at 100,000 times g.


RESULTS

When mouse meprin alpha subunits were expressed in COS-1 cells, most of the immunoreactive material was found in the tissue culture medium as secreted protein with a subunit mass of about 95 kDa (Fig. 1, topgel, lane3). The secreted protein was catalytically inactive, but could be activated with trypsin treatment (data not shown). A small fraction of the expressed meprin alpha subunits was found associated with cellular membranes (Fig. 1, topgel, lane4); the cell-associated meprin consisted of several bands with apparent molecular masses of 86-102 kDa, with the 86 kDa band being the predominant form. The supernatant fractions of cell homogenates did not contain any immunoreactive material (data not shown). Under nonreducing conditions, the secreted and the cell-associated meprin subunits were oligomeric with apparent molecular weights larger than 180,000 (Fig. 1, top gel, lanes 5 and 6). The cell-associated meprin subunits were sensitive to Endo F and Endo H, indicating that complex glycosylation had not yet occurred (Fig. 1, bottomgel). Treatment with either of the endoglycosidases produced two bands with molecular masses of approximately 81 and 65 kDa (Fig. 1, bottomgel, lanes1-3), with the 65 kDa band being the predominant form. In contrast, the secreted meprin subunits in the culture medium contained some carbohydrate chains that are resistant to Endo H treatment (Fig. 1, bottomgel, lanes4-6). Endo F treatment decreased the molecular mass of the secreted meprin alpha subunit from approximately 95 to 67 kDa; Endo H treatment decreased the molecular mass to approximately 79 kDa. Thus, in transfected COS-1 cells, the mature mouse alpha subunit is primarily a secreted protein. A small amount of meprin subunits remains associated with the endoplasmic reticulum (ER), indicated by the Endo H sensitivity. There are no detectable amounts expressed on the cell surface because no Endo H-resistant forms of meprin were detected associated with cells. These results are generally consistent with those obtained for the transfected human and rat meprin alpha subunits in COS-1 cells(12, 13, 15) . Furthermore, the molecular mass of the predominant cell-associated deglycosylated mouse subunit indicated that COOH-terminal processing occurs in the ER.


Figure 1: Immunoblot analysis of the mouse meprin alpha subunit expressed in COS-1 cells. COS-1 cells were transfected with wild-type meprin alpha subunit cDNA as described under ``Experimental Procedures.'' Concentrated tissue culture media (medium) and membrane-enriched fractions (cells) were subjected to SDS-PAGE and immunoblotting using anti-meprin alpha subunit antibodies. Topgel, SDS-PAGE in the presence or absence of beta-mercaptoethanol. Lanes1 and 2 contained control samples transfected with vector DNA only; lanes3-6 contained samples after transfection with meprin alpha subunit cDNA. Bottomgel, SDS-PAGE in the presence of beta-mercaptoethanol and immunoblot analysis of membrane-enriched fractions or media samples. Samples were incubated with SDS and beta-mercaptoethanol in the presence or absence of Endo F or with Endo H. The relative positions of the molecular mass markers (in kDa) are shown.



To determine whether the I domain of alpha is necessary for COOH-terminal processing, two mutant meprin subunits lacking this domain were expressed in COS-1 cells (Fig. 2). In construct alphaDelta628-683, the I domain of alpha was deleted by site-directed mutagenesis. In construct alpha/beta, the alpha subunit domains COOH-terminal to the X domain were replaced with the corresponding domains of the beta subunit. Analysis of the culture media and membrane fractions by immunoblotting showed that, in contrast to the wild-type alpha subunit, neither of the mutant meprin subunits were secreted into the culture medium, they were found exclusively in membrane fractions (Fig. 2, panelA). In addition, the molecular masses of the cell-associated mutant subunits were slightly larger than the molecular masses of the secreted wild-type alpha subunit (approximately 105 kDa for the alpha/beta chimeric subunit and 102 kDa for the alphaDelta628-683 mutant, compared with 95 kDa for the wild-type secreted alpha subunit). The larger molecular masses of the mutant subunits compared with the wild-type subunit provide further evidence that these mutants are not proteolytically processed at the COOH terminus but retain the EGF-like and transmembrane domains. To test whether the mutant proteins are expressed on the cell surface or retained intracellularly, the isolated membranes were subjected to endoglycosidase digestion (Fig. 2, panelB). The alphaDelta628-683 mutant was Endo H-sensitive, and thus was retained in a preGolgi compartment. In contrast, the alpha/beta chimeric subunit was resistant to Endo H digestion, indicating that this subunit was present in a postGolgi compartment, most likely the cell surface. The results of transfecting the alpha/beta chimeric subunit indicate that the COOH terminus of the alpha subunit, but not the beta subunit, contains the information for COOH-terminal processing. The alphaDelta628-683 mutant was not processed and not able to leave the ER, indicating the importance of the I domain for processing and secretion.

To further test whether the COOH-terminal removal of the membrane anchor of alpha occurs in the ER, carbonate treatment of isolated membranes from transfected COS-1 cells was performed. Carbonate treatment releases lumenal proteins and peripheral membrane proteins from membrane vesicles, while transmembrane proteins remain associated with the membrane vesicles and thus precipitate during centrifugation (27) . Carbonate treatment of membranes from cells transfected with wild-type alpha subunits solubilized at least 50% of meprin alpha subunit protein but failed to solubilize any meprin protein from cells transfected with the alphaDelta628-683 mutant (Fig. 2, panelC). Thus, ER-associated wild-type meprin alpha subunits are lumenal/peripheral ER-associated proteins that lack the COOH-terminal membrane-anchoring domain, while the alphaDelta628-683 mutant is membrane-anchored. These data provide further evidence that COOH-terminal processing of the membrane anchoring domain occurs early in meprin biosynthesis, before the acquisition of complex carbohydrate modifications, and that the I domain is essential for proteolytic processing.

To test whether the I domain is sufficient to determine meprin subunit processing, the alpha subunit I domain was inserted in-frame into the rat beta subunit sequence. For this experiment the rat beta subunit, which is more than 90% identical to the mouse subunit, was used (rather than the mouse beta subunit) because of the availability of a specific and sensitive antiserum to the rat beta protein. The insertion of the I domain was achieved in two steps; in the first step, the COOH-terminal domains of beta (EGF-like, transmembrane, and cytoplasmic) were replaced with the alpha subunit COOH-terminal domains (I, EGF-like, transmembrane, and cytoplasmic) (construct beta/alpha). In the second step, the EGF-like, transmembrane, and cytoplasmic domains of the beta/alpha construct were replaced with those of beta (construct beta/alpha/beta). After expression in COS-1 cells, wild-type rat beta subunits were predominantly membrane-bound as previously reported for rat and human beta subunits. In contrast, both mutants beta/alpha and beta/alpha/beta were predominantly secreted (Fig. 3). Thus, insertion of the alpha subunit I domain into the beta subunit sequence is sufficient to direct COOH-terminal proteolytic processing and secretion of normally membrane-bound beta subunits.

The I domain of alpha contains two dibasic sites, amino acids Arg/Lys and Lys/Arg that could serve as processing sites by enzymes with specificity for dibasic motifs. Amino acids Lys/Arg are also part of the consensus sequence for cleavage by furin-like processing enzymes (RX(K/R)R)(17) . To test whether the furin-like consensus sequence or the other dibasic site are cleavage sites in alpha, the dibasic sites were replaced by glycine residues, and the resulting mutant proteins were expressed in COS-1 cells and analyzed for secretion and cellular localization (Fig. 4, leftgel). Eliminating either or both of the two dibasic sites did not prevent secretion of the meprin alpha subunit into the culture medium, indicating that neither of these sites is essential for processing.

In an attempt to map the cleavage site to a smaller region within the inserted domain, portions of the inserted domain were deleted (constructs alphaDelta628-660 and alphaDelta661-683), and the resulting mutants were analyzed (Fig. 4, rightgel). Both mutants were processed and secreted into the culture medium. The apparent molecular mass of alphaDelta628-660 seemed slightly larger than the molecular mass of the wild-type or alphaDelta661-683 protein; however, after deglycosylation with Endo F, all three proteins had indistinguishable molecular masses (data not shown). The mutant alphaDelta661-683 was secreted in amounts comparable with wild-type alpha subunits, while the alphaDelta628-660 construct was secreted at somewhat lower levels. These results indicate that the entire 56-amino acid sequence of the I domain is not required for processing and that, if the processing site is located within the inserted domain, more than one peptide bond can be cleaved by the processing machinery.

To determine whether the COOH-terminal domains in alpha are important during meprin biosynthesis for either dimerization or transport of meprins through the secretory pathway, several COOH-terminal deletions were constructed and expressed in COS-1 cells (Fig. 5). Up to 133 amino acids, including the I domain, were deleted from the alpha subunit COOH terminus. All truncated proteins were secreted, and none of the deletions affected the level of protein biosynthesis or the ability of subunits to dimerize.

Pulse-chase experiments in 293 cells showed that mouse meprin alpha subunits were present in two major forms (95 and 86 kDa) after 10 min of exposure to [S]methionine and that with time, the 95-kDa form was converted to the 86-kDa form (Fig. 6). By 30 min, most of the radiolabeled subunits were of the 86-kDa form, and a small amount of this form persisted in cells up to 6 h. The cell-associated forms of the alpha subunit were Endo H-sensitive for all time points (data not shown). Immunoprecipitable radiolabeled alpha subunits were detected in culture media by 1 h; secreted forms of the subunit were Endo H-resistant as shown in Fig. 1. Thus, the pulse-chase experiments indicated that two major glycosylated forms of alpha are produced early in biosynthesis (about 15-30 min), and that there is a conversion from the larger to the smaller form before the meprin alpha subunits acquire complex glycosylation.


Figure 6: Pulse-chase radiolabeling of transfected 293 cells expressing wild-type mouse alpha meprin subunits. Cells were radiolabeled with [S]methionine for 10 min and then incubated in nonradioactive methionine-containing medium for the times shown. At each time point, cells were harvested and lysed, and the cells and medium were prepared as described under ``Experimental Procedures''. Meprin alpha subunits were immunoprecipitated with an anti-meprin alpha subunit antibody. The proteins were subjected to SDS-PAGE in the presence of beta-mercaptoethanol and visualized by fluorography.



In order to determine more definitely whether the processing observed in pulse-chase experiments was due to COOH-terminal proteolytic processing, a rat meprin alpha cDNA with a c-myc epitope at the COOH terminus of the subunit was transfected into 293 cells. The disappearance of the c-myc tag was studied by radiolabeling the transfected cells with [S]methionine (Fig. 7). A monoclonal antibody against the c-myc epitope only immunoprecipitated the largest form of radiolabeled alpha subunits. The anti-alpha meprin subunit antibody immunoprecipitated several smaller forms of the subunit in addition to the COOH-terminally unprocessed form. Furthermore, all forms associated with cells were Endo H-sensitive, indicating that they exist in the ER. The results of this experiment confirm that the COOH terminus of the meprin alpha subunit is proteolytically processed in the ER.


Figure 7: Immunoprecipitation of transfected meprin alpha subunits containing a c-myc epitope tag at the COOH terminus, with anti-meprin alpha subunit antibodies or anti-c-myc antibodies. Human 293 cells were transfected with expression vector (control) or expression vector containing the rat meprin alpha cDNA tagged with a c-myc epitope at the COOH terminus (c-myc). The cells were radiolabeled with 0.1 mCi/ml [S]methionine for 1 h. Radiolabeled meprin alpha subunits were then immunoprecipitated from cell lysates, subjected to SDS-PAGE, and visualized by fluorography. Samples in lanes1-3, 6, and 7 were immunoprecipitated with anti-meprin alpha subunit antibodies. Samples in lanes4 and 5 were immunoprecipitated with anti-c-myc antibodies. For lanes2 and 3, immunoprecipitated cellular proteins were treated with Endo H and F, respectively, prior to electrophoresis.




DISCUSSION

It is clear from the results presented herein that several important events in the maturation of meprin subunits occur in the ER (shown diagramatically in Fig. 8). The experiments indicate that subunits are translated, dimerize, form intersubunit disulfide bridges, and are glycosylated (high mannose), and, if the I domain is present, the domains COOH-terminal to the X domain are proteolytically removed in the ER. Once all these events occur, the subunits appear to exit the ER, obtain complex sugars (probably in the Golgi apparatus), and are secreted from cells through the constitutive pathway. This was the scenario for most of the transcripts expressed herein in the cultured cells, including wild-type alpha subunits (Fig. 8, panelA), alpha subunits truncated down to the X domain, alpha subunits with the dibasic sites of the I domain mutated to GG, alpha subunits with the COOH-terminal or NH(2)-terminal halves of the I domain deleted, and beta subunits with the I domain inserted between the X and EGF-like domains. There was very little evidence of monomeric or nonglycosylated forms of these proteins in the cells, indicating that dimerization and high-mannose glycosylation take place immediately after translation. This could be important for the stabilization of the subunits. Many proteins are degraded in the ER if not properly assembled, and the oligomerization and glycosylation of the subunits might protect the subunits from destruction in the ER, as was shown for proteins such as the T cell receptor subunits, class II major histocompatibility complex molecules, and the transferrin receptor(28, 29, 30) . In addition, there was no evidence for complex glycosylated meprin alpha subunits in the transfected cells, which indicates that once the subunits leave the ER they are secreted rapidly, without much delay in other subcellular compartments.


Figure 8: Diagrammatic representation of the biosynthesis of meprin subunits. Meprin subunits transfected into COS-1 cells were either secreted (A), transported to the cell surface as membrane-bound proteins (B), or retained in the ER as membrane-bound proteins (C). The alpha subunit sequences are indicated by openstructures; beta subunit sequences are indicated by shadedstructures. The I domain is indicated in panelA as a loop between the EGF-like domain (oval) and the main body of the subunits which includes the X, MAM, and protease domains (rectangles); the I domain is not present in transcripts of panelB and C. Core N-linked glycosylation is indicated for all nascent proteins by Y-shaped structures perpendicular to the subunits. Complex glycosylation is indicated by circles on the Y-shaped structures.



A different final localization was observed for wild-type beta subunits (Fig. 8B) and the alpha/beta chimera without the I domain. These subunits also dimerize, leave the ER, and enter the Golgi where they obtain complex glycosylation. Thus, they probably follow the constitutive pathway to the cell surface, as the secreted forms. However, the lack of the I domain in these subunits prevents their COOH-terminal processing, and as a consequence they remain membrane-bound after they reach the cell surface.

A third pathway was observed only for alpha subunits with the I domain deleted (Fig. 8C). These subunits accumulate as unproteolyzed high mannose precursors in the ER without being further transported to the Golgi. Thus, the I domain is essential for processing. The data further indicate that the COOH-terminal domains of alpha contain information that leads to retention in the ER. It is clear that the I domain in itself is not essential for intracellular transport because COOH-terminally truncated subunits lacking the I domain are secreted from cells. Rather, it is more likely that the deletion of the I domain causes the accumulation in the ER by preventing the proteolytic removal of an ER retention determinant COOH-terminal to the I domain. The retained alpha subunit with a deleted I domain differs from the alpha/beta chimeric subunit, which is not retained, only in that the COOH-terminal EGF, T, and C domains are derived from alpha instead of beta; thus these COOH-terminal domains determine retention or movement out of the ER. It is probable that determining factors for retention lie in the T or C domains of alpha. The transmembrane and cytoplasmic domains of alpha and beta share no significant homology, in contrast to the rest of the subunit protein. There is a growing number of membrane proteins, in which ER retention signals have been identified in the transmembrane or cytoplasmic domains(31, 32) . These signals are distinct from the tetrapeptide KDEL-like sequences at the COOH termini of lumenal ER proteins, which determine retention through a receptor-mediated retrieval pathway(33) . The T domain of alpha contains a motif of repeating glycines, also found in class II major histocompatibility complex molecules and glycophorin, which have been proposed to form a surface that can mediate interactions with other transmembrane helices(29, 34) . These repeating glycines are not present in the beta T domain. The retention of unprocessed alpha subunits ensures that alpha subunits only exit the ER after COOH-terminal processing, and may be an example of the quality control function of this subcellular compartment(35) .

The results herein demonstrate that the I domain is essential for processing. However, the attributes of this domain that are required for processing are not yet defined. It cannot be completely ruled out that the I domain determines processing at a nearby sequence in alpha through conformational effects. However, the observation that the I domain can function as a processing domain in the context of a different protein (the beta subunit) makes it more likely that the processing site(s) lies within the I domain itself. This is also consistent with previous calculations of the molecular size of processed deglycosylated alpha subunits from mouse kidney, which indicated that the mature alpha subunit ends in the NH(2)-terminal part of the I domain(11) . The simple possibilities of the furin site or dibasic site, or both sites being essential sites for cleavage were eliminated by the site-directed mutagenesis experiments. Deletion of the NH(2)- or COOH-terminal halves of the I domain also did not prevent processing of the subunits, as they were still predominantly secreted. Thus, more than one cleavage site can be used by the processing enzyme(s). It is possible that the processing site is not a specific recognition sequence but that processing occurs at an exposed region that is vulnerable to proteolysis. The I domain might be a region of poorly defined secondary or tertiary structure such as a surface exposed loop, which is susceptible to cleavage by resident ER proteases that are known to degrade misfolded proteins.

Dimerization is clearly not dependent on the T domain, as with some other proteins such as class II major histocompatibility complex molecules, glycophorin, and hemagglutinin, nor does it depend on the EGF-like or C domains because deletion of all of these still results in dimers(29, 34, 36) . Perhaps the MAM or X domains are important for alignment of the subunits and intersubunit disulfide bridge formation.

It is well established that the proteolytic system in the ER is responsible for the total degradation of many misfolded or incompletely assembled proteins, as well as resident ER proteins(28, 37, 38, 39, 40) . There is also some indication that processing for antigen presentation can occur in the ER(41) . Known instances of limited proteolysis in the ER, as seen with the meprin alpha subunit, are rare, with the exception of signal peptide cleavage by signal peptidase(42, 43) . The specific limited protein processing described in the present work may constitute a new function for ER proteases. Autocatalytic processing of some proteases, e.g. kexin, occurs in the ER(44) , but it is unlikely that the processing of meprin alpha is due to self-cleavage because the subunits are synthesized as inactive zymogens in COS-1 and 293 cells. Proteases that have been implicated in ER degradation are cysteine proteases, e.g. ER-60 and ER-p72(39, 45, 46) , and a serine protease(37) . Meprins provide a good system for investigating and identifying the ER proteases involved in the limited proteolysis function of this subcellular compartment.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant DK 19691. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Penn State University, Hershey, PA 17033. Tel.: 717-531-8586; Fax: 717-531-7072.

(^1)
The abbreviations used are: EGF, epidermal growth factor; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PMSF, phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; Endo F, endoglycosidase F and N-glycosidase F; Endo H, endoglycosidase H; ER, endoplasmic reticulum.


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