The Primary Structure of Glycoprotein III from Bovine Adrenal Medullary Chromaffin Granules SEQUENCE SIMILARITY WITH HUMAN SERUM PROTEIN-40,40 AND RAT SERTOLI CELL GLYCOPROTEIN

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secre- tory vesicles of the adrenal medulla, by chromatogra- phy using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatog- raphy (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis, and partial NHa-terminal sequence analysis indicated that GpIII was a disulfide-linked heterodimer with 37- kDa subunits. Analysis of in vitro translation products of medullary itation using


Glycoprotein
III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis, and partial NHa-terminal sequence analysis indicated that GpIII was a disulfide-linked heterodimer with 37-kDa subunits.
Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor.
Partial NHz-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has >80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.
Chromaffin granules are subcellular organelles of the adrenal medulla with specialized functions for the biosynthesis, storage, and secretion of catecholamines and some peptides and proteins, such as enkephalins and chromogranins. These vesicles are relatively easy to purify in good yield and have been used extensively as model secretory granules. Considerable information is available on the composition of these vesicles (see Ref. 1 for a review).
Most of the information available regarding chromaffin granule membrane proteins is based on electrophoretic techniques. Over 40 membrane proteins have been identified (2) many of which are glycosylated. Five major membrane glycoproteins, termed Gp'I-V, have been identified by lectin binding (3). Subsequently, improvements in analysis using two-dimensional gel electrophoresis with lectin binding and membrane fractionation using Triton X-114 have enabled the identification of at least 20 membrane glycoproteins (4, 5). While the composition of chromaffin granule membranes has been increasingly well studied there is little detailed structural information available for the membrane proteins. Three glycoproteins, GpI (dopamine P-hydroxylase) GpII, and GpIII, have been purified from chromaffin granule membranes using sequential lectin affinity chromatography (6). These workers determined that GpIII had a high carbohydrate content (32%), with the major sugars being galactose, Nacetylglucosamine, sialic acid, and mannose. The carbohydrate moieties are exposed in the inner or matrix side of the chromaffin granule membrane (3). GpIII was later shown to also be a component of the soluble contents of chromaffin granules (7). No differences were found between the membrane and soluble forms of GpIII. Both are present as acidic glycoproteins which migrate in Laemmli gels as proteins of relative molecular mass 74,000 under non-reducing conditions and 37,000 under reducing conditions (7).
There are a number of reasons which make GpIII interesting. An antiserum raised against this glycoprotein has been used to show that the chromaffin granule membrane is returned to the Golgi following exocytosis and recycled into new secretory granules (8). It has been demonstrated immunologically that GpIII is also present in pituitary tissue (7), suggesting that GpIII may have a general role in secretory vesicles. Finally, GpIII, like dopamine P-hydroxylase and carboxypeptidase H, has membrane and soluble forms for which neither the structural basis or functional significance is known (9-11).
In the present paper we report a purification and characterization of soluble GpIII. The complete amino acid sequence has been deduced from cloned cDNA. GpIII was found to have sequence similarity to human serum protein-40,40 (12), a glycoprotein associated with complement components, and rat Sertoli cell glycoprotein 2 (13), a glycoprotein which may be important in spermatogenesis. GpIII revealed two sequences in approximately equal yield. When GpIII was reduced and alkylated and subjected to reverse-phase HPLC, two major peaks were isolated (Fig. 2B). The NH1-terminal sequence of the earlier eluting peak, termed the A chain, was Ile-Ser-Asp-Lys-Glu-Leu-Gln-Glu-Met-Ser-Thr-Glu-Gly and the NH?-terminal sequence of the later eluting peak, termed the B chain, was Asn-Val-Met-Pro-Phe-Pro-Leu-Leu-Glu-Pro-Phe-Asn-Phe-His-Asp-Val-Phe-Gln-Pro.
The two sequences agreed with the results obtained with GpIII prior to reduction. Adrenal medullary poly(A)+ RNA (1 rg) was translated in vitro in the presence of ["YS]methionine. The translation products were subjected to immunoprecipitation using an anti-GpIII serum in the absence (lane 1) or presence (lane 2) of added non-radioactive GpIII (approximately 1 pg). The immunoprecipitated products were subjected to SDS-PAGE, using a 12% gel, followed by fluorography.

Analysis of the isolated A and B chains
by SDS-PAGE showed that they have a similar relative molecular mass (Fig.  1). Both chains contain carbohydrate which can be removed with glycopeptidase F (data not shown). Presumably, microheterogeneity within the carbohydrate moieties gives rise to the appearance of GpIII in SDS-PAGE as a broad band. The amino acid composition of the two chains, while not identical, was similar (data not shown). The A and B chains were compared further by tryptic digestion and peptide mapping by reverse-phase HPLC (data not shown). The two chains appeared to have no tryptic peptides in common, indicating that GpIII is a heterodimer containing disulfide-linked subunits. Immunization of rabbits with purified GpIII gave an antiserum which recognized the isolated A and B chains before and after treatment with glycopeptidase F (data not shown). Adrenal medullary poly(A)+ RNA was translated in vitro, and an immunoprecipitation was carried out using the anti-GpIII serum. Fig. 3 shows that a single translation product was specifically immunoprecipitated, suggesting that the A and B chains are derived from a single precursor.
Isolation and Characterization of a GpIII-specific cDNA Clone-A DNA probe to GpIII was generated by mixed oligonucleotide-primed amplification of cDNA (24). The primers were based on the NH2-terminal sequence of the B chain and an internal sequence obtained from a cyanogen bromide fragment of this chain (Fig. 4A). These primers were used to amplify single-stranded cDNA prepared from adrenal medullary poly(A)+ RNA. After 25 cycles of amplification a single DNA product of approximately 650 bp was obtained (Fig.   4B).
When the PCR product was used as a probe for a Northern blot analysis of total adrenal medullary RNA, a single RNA species of approximately 2000 bp was detected (Fig. 4C).
A cDNA library was constructed in XgtlO from adrenal medullary poly(A)+ RNA and screened using the PCR product A, the NH?-terminal sequences of the B chain (sense primer) and a CNBr fragment of the B chain (antisense primer) are shown with the regions used for primer construction underlined.
The primers are shown below the corresponding amino acid sequences with the restriction sites underlined.
An asterisk indicates positions where not all of the possible codons are represented. An NHp-terminal methionine (') was assumed. B, the products of the PCR (i/20) were analyzed after 25 cycles by electrophoresis on a 1.5% agarose gel followed by staining with ethidium bromide (lane 2). Lane I contains size markers (Bethesda Research Laboratories). C, Northern blot analysis of total adrenal medullary RNA (25 pg). The RNA was fractionated on a 1.4% agarose gel and the nitrocellulose blot was hybridized with the radioactively labeled PCR product. by guest on March 22, 2020 http://www.jbc.org/ Downloaded from as probe. Forty positive clones were detected from approximately 20,000 plaques. A number of the clones were plaquepurified and characterized by restriction mapping. The longest clone contained a 1700-bp insert which was recovered as three EcoRI fragments which were cloned into Bluescript and sequenced, as outlined in Fig. 5A. The alignment of the three EcoRI fragments was deduced from the sequence obtained from the cloned PCR product. Fig. 5B shows the nucleotide sequence of the cDNA. The predicted amino acid sequence of the GpIII precursor is also shown in Fig. 5B and appears to be complete. The deduced sequence is in complete agreement with amino acid sequence results, containing the regions corresponding to the NH*-terminal sequence of the A and B chains as well as NH*-terminal sequences obtained from CNBr fragments.
We were surprised to find that the sequence of GpIII was A similar to that found for human serum protein-40,40 (12) and rat Sertoli cell glycoprotein 2 (13). An alignment of the amino acid sequences of these proteins is shown in Fig. 6. Northern blot analysis of RNA prepared from a range of bovine tissues showed that cDNA encoding GpIII hybridized with RNA species of similar size in the adrenal medulla, liver, testis, brain, and spleen (Fig. 7). In testis, a larger and less abundant RNA species was also detected.

DISCUSSION
In this paper we describe a purification and characterization of GpIII from bovine adrenal medulla. Several factors were found to have a major influence on the final purity and yield of this glycoprotein.
The purity of GpIII following WGA-Sepharose chromatography is greatest when a near saturating A, a partial restriction map and sequencing strategy for the cDNA clone. All sequencing was performed in Bluescript using the T7 and T3 primers except for one sequence which was obtained using a GpIII-specific primer (1). The information required to align the three EcoRI fragments of the cDNA was obtained from the sequence of the cloned PCR product (2). B, nucleotide sequence of the cDNA and predicted amino acid sequence of the GpIII precursor. Regions of the predicted amino acid sequence corresponding to GpIII amino acid sequence determined by protein analyses are underlined.
Arrowheads indicate the sites of proteolytic processing which would give rise to the NHz-terminal sequences of the A and B chains. The putative signal peptide is designated -19 to -1. Potential N-linked glycosylation sites are indicated (0). An asterisk indicates the stop codon. Identities are indicated with dashes. Gaps are indicated with dots. The numbering refers to the GpIII sequence. Potential N-linked glycosylation sites are shown for GpIII (0). Sites of proteolytic processing are indicated for GpIII above the sequences and for SP-40,40 and SGPZ, which are identical, below the sequences, by arrowheads. Total RNA (25 pg) isolated from bovine adrenal medulla (lane I), liver (lane 2), testis (lane 31, brain (lane 41, and spleen, (lane 5) was fractionated on a 1.4% formaldehyde-agarose gel, and the nitrocellulose blot was hybridized with radioactively labeled GpIII cDNA present in Bluescript. No hybridization was detected when Bluescript DNA alone was used as a probe (not shown). amount of GpIII is applied to the column. Attempts to purify GpIII from smaller amounts of lysate resulted in an increased relative amount of other proteins, particularly dopamine phydroxylase in the N-acetylglucosamine eluate. Presumably under these conditions GpIII, which has a relatively high affinity for WGA, displaces more weakly bound proteins from the column. In addition, the recovery of GpIII from HPLC was best when the concentrated WGA-Sepharose eluate was applied to the column at neutral pH. Acidification of the sample resulted in a dramatically decreased yield, presumably because GpIII like secretogranin II, a pituitary secretory granule protein aggregates at low pH (25). To avoid losses, peaks collected from HPLC, containing GpIII, were neutralized immediately.
The predicted amino acid sequence of GpIII precursor confirms the results of earlier protein characterization. The sequences of the A and B chains are contained within a single precursor. The amino acid residues at -3 and -1 suggest a cleavage site for signal peptidase between -1 and +1 (26). Such a cleavage would result in an NH2-terminal sequence which corresponds to the NH*-terminal sequence of the A chain. An additional proteolytic cleavage at an Arg-Asn (position 202-203) would generate an NH*-terminal sequence identical to the NH*-terminal sequence of the B-chain and form the disulfide-linked dimer.
GpIII contains approximately 30% carbohydrate (6) which is consistent with the difference between the calculated protein molecular masses of the A chain (23,620 Da) and the B chain (25,335 Da) and the relative molecular masses of the mature subunits (37,000). Both subunits contain carbohydrate, all of which appears to be N-linked since the relative molecular masses of the subunits following treatment with glycopeptidase F correspond to the calculated protein molecular masses of the two chains (data not shown). There are three potential N-linked glycosylation sites in the predicted sequence of the A chain and five in the B chain. Analysis of partially deglycosylated subunits using glycopeptidase F suggests that most if not all of the potential N-linked glycosylation sites in each chain are utilized.* The calculated molecular mass of the GpIII precursor (51,100 Da) does not agree with the relative molecular mass (45,000) of the immunoprecipitated in uitro translation product although the reason for this discrepancy is not known.
It is known that GpIII is present in chromaffin granules in both a membrane-bound and soluble form (6, 7). The membrane-associated species behaves as an integral membrane protein, requiring high concentrations of detergent to affect solubilization (6) and is prone to aggregation.* Several possible mechanisms of membrane attachment appear unlikely. The predicted amino acid sequence of GpIII contains no obvious hydrophobic segment other than the leader peptide which is capable of transversing the membrane. Anchorage by an uncleaved leader peptide can be excluded since NHp-terminal sequence analysis of the membrane-bound form of GpIII revealed the same two NH*-terminal sequences obtained with the soluble form.* It is also of interest to note that the unreduced membrane-bound and soluble forms of GpIII elute at the same position from HPLC and that they both bind detergent when analyzed by charge-shift electrophoresis.* It is possible that a conformational change may convert GpIII from a soluble to a membrane-associated species as has been established for complement protein C9 (27) quence and a covalent glycosyl phosphatidylinositol anchor have recently been excluded as possibilities for dopamine phydroxylase (28,29). When we began this work it seemed likely that GpIII was a secretory granule component specific to endocrine tissues as it has been detected using immunological methods in anterior and posterior pituitary but not in liver, pancreas, or parotid gland (7). It was therefore surprising to find that GpIII shared a high degree of sequence similarity with a human serum glycoprotein, 40 (12), and a rat Sertoli cell glycoprotein, SGP2 (13) also termed clusterin. Comparison of the amino acid sequence of GpIII with SP-40,40 revealed a 72% level of identity and with SGP2 a 67% level of identity, suggesting that these proteins are species counterparts. In support of this, identical RNA species were detected by Northern blot analysis in adrenal, liver and testis.
Alignment of the amino acid sequences of GpIII, SP-40,40, and SGP2 (Fig. 6) shows a number of interesting features. While the sequence identity is 61%, in some regions and in particular two cysteine-rich motifs (residues 77-104 and 25& 286) the identity is striking. The first of these regions (residues 77-104) is related to sequences found in several terminal complement components (12) and is likely to be an important structural feature of these proteins. GpIII contains eight potential N-linked glycosylation sites whereas SP-40,40 and SGP2 each contain six, all of which correspond to potential sites in GpIII. The three proteins differ in the length of the leader sequences and at the NH, termini of the subunits equivalent to the A chain of GpIII. The two-chain structure of the mature glycoproteins however, results from cleavage at a similar site in all three proproteins.
It is difficult to compare the physiological functions of GpIII, SGPP, and SP-40,40. SGP2 is a major secretory product of rat Sertoli cells and is identical to rat and equivalent to ram clusterin, so named because of their cell aggregating activity in uitro (30,31). It is interesting to note that another adrenal medullary protein, proenkephalin is also present in the testis and secreted by Sertoli cells (32,33). Human SP-40,40 was discovered in association with terminal complement components in immune deposits in a patient suffering from glomerular nephritis, and it is also found associated with the soluble variant of the C5b-9 complex (34). In a recent study, a separate group reported the cDNA sequence of a human protein, identical to SP-40,40, which was shown to function as an inhibitor of terminal complement component-dependent cytolysis (35). Evidence was also presented that these proteins are encoded by single-copy genes in rats and humans.
Comparison of the results presented in this paper with work on SGP2 and SP-40,40 indicates that GpIII has a wide tissue distribution. The RNA encoding this glycoprotein appears to be relatively abundant in adrenal medulla, anterior and posterior pituitary, brain, liver, and testis and is also present in spleen, kidney, and mammary tissue. It is likely that the liver represents the major source of the serum form of this protein.
There is some evidence to suggest that post-translational modifications, particularly glycosylation, to GpIII are not identical in all tissues. The relative molecular masses of clusterin from ram serum and testis are not identical, which appears to be due to dissimilar glycosylation (36). Furthermore, ram testis clusterin is capable of agglutinating red blood cells in uitro, whereas this activity is not observed with ram serum clusterin. The agglutinating activity of ram testis clusterin can be abolished by chemical or enzymatic deglycosylation (36). We were unable to observe agglutination of red blood cells with GpIII. ' In the adrenal medulla, GpIII represents a secretory product sorted to secretory vesicles and (in the case of the soluble form) released in response to acetylcholine. It is likely that the equivalent protein is constitutively expressed in liver and possibly Sertoli cells where some of the single chain precursor is also known to be secreted (13). It would therefore be of interest to determine the sorting and secretion of this protein in a cell line such as mouse AtT-20 cells which have well characterized constitutive and regulated pathways (37). One common feature of GpIII, SGPe/clusterin, and SP-40,40 is their hydrophobicity, exhibited by their retention on reverse-phase HPLC, susceptability to aggregation, ability to bind to cells, membranes, and hydrophobic proteins. It is possible that these properties may contribute toward a function in a variety of tissues. In this respect it will be important to understand the molecular basis for the presence of both soluble and membrane forms of this protein in chromaffin granules.