Zinc-α2-Glycoprotein Has Ribonuclease Activity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 355, No. 2, July 15, pp. 160 –164, 1998 Article No. BB980735

Zinc-a2-Glycoprotein Has Ribonuclease Activity Gang Lei,*,† Istvan Arany,† Stephen K. Tyring,*,† Henry Brysk,* and Miriam M. Brysk*,†,‡,1 *Department of Dermatology, †Department of Microbiology and Immunology, and ‡Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555

Received January 15, 1998, and in revised form April 29, 1998

Zinc-a2-glycoprotein (Zna2gp) is widely distributed in body fluids and in various epithelia; its gene has been completely sequenced, but its function has long remained elusive. We have found that Zna2gp has RNase activity, comparable to onconase but two orders of magnitude less than RNase A. The RNase activity of Zna2gp is characterized by maxima in pH at 7.5, in ionic strength at 50 mM NaCl, and in temperature at 60°C. It is strongly inhibited by ZnCl2, but unaffected by MgCl2. It is partially inactivated (down to 20%) by the placental RNase inhibitor. On synthetic polyribonucleotide substrates, the RNase activity of Zna2gp is specific for pyrimidine residues [poly(C) and poly(U) equally] and cleaves only single-stranded RNA. For onconase, it has been demonstrated that the RNase activity depends on pyroglutamic acid (pyr 1) as the N-terminus; Zna2gp also has pyr 1, while RNase A does not. Alignment of the amino acid sequences of Zna2gp and onconase or RNase A reveals only modest matches. Despite the more substantial overall structural homology of Zna2gp to class I major histocompatibility complex proteins, Zna2gp has not been proven to be associated with the immune response and, conversely, we could not detect RNase activity in six class I HLA heavy chains. © 1998 Academic Press Key Words: zinc-a2-glycoprotein; RNase activity; MHC; onconase.

Zinc-a2-glycoprotein is a soluble protein initially purified from plasma (1); it can be precipitated by adding zinc ions and it displays electrophoretic mobility in the a2-region of the plasma globulins, hence its name. It has been detected in most body fluids, including blood, seminal plasma, breast milk, synovial fluid, saliva, 1

To whom correspondence should be addressed at Department of Dermatology, University of Texas Medical Branch, Galveston, TX 77555-0783. Fax: 1-409-772-1943. 160

urine, and sweat (2–5). Its antibody labels a wide variety of secretory epithelia in various human glands (6). Northern blot analyses reveal the gene to be expressed in liver, breast, prostate, kidney, pancreas, and several tumors (7, 8). Zna2gp has been cloned and its complete genomic sequence determined for prostate (9) and breast (10); the cDNA sequences are ascribed to a single active gene and one or two pseudogenes. The corresponding amino acid sequences appear to be similar for these two tissues, as also for blood plasma (11), consisting of a single polypeptide chain of 278 amino acids. Glycosylation varies among tissues, mostly about 12–18% carbohydrate except that seminal plasma is unglycosylated (4). The molecular weight is in the range 38 – 41 kDa, depending on the tissue. We have found that Zna2gp is also expressed in stratified epithelia; in particular, we have cloned it from epidermal keratinocytes and found a nucleotide sequence identical to that from prostate (12), differing only in posttranslational modifications. The amino acid sequence of Zna2gp has 36 –39% homology to major histocompatibility complex (MHC)2 class I antigens, although Zna2gp, as a soluble protein, lacks the coding information for transmembrane and cytoplasmic domains (11). There is an analogous homology at the nucleotide level (56% identity to HLAB7) (8). Nonetheless, there is no evidence that Zna2gp is involved in the immune response. The gene for Zna2gp has been localized to chromosome 7 as against chromosome 6 for MHC (9, 13). Zna2gp differs from class I MHC in its lack of polymorphism (14) and does not bind peptides from the class I light chain, b2-microglobulin (15). Despite its widespread occurrence in different body tissues and extensive studies of its molecular, protein, and crystalline structure (15), Zna2gp continues to be described in the literature as a protein of unknown function. A significant association has been observed 2

Abbreviation used: MHC, major histocompatibility complex. 0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

ZINC-a2-GLYCOPROTEIN HAS RIBONUCLEASE ACTIVITY

between Zna2gp levels and the histological grade of breast cancer tumors, with higher levels found in welldifferentiated tumors than in moderately or poorly differentiated ones, by protein assays (14) as well as at the mRNA level (8). Analogously, Zna2gp levels are much higher in benign prostatic hyperplasia than in adenocarcinoma of the prostate (3) and Zna2gp gene expression is associated with differentiation in oral squamous cell carcinomas (16). The only previously reported activity of Zna2gp is the promotion of cell adhesion and spreading in culture, selectively for several cell lines but not others (17). MATERIALS AND METHODS Substrates and reagents. Synthetic polynucleotides [poly(C), poly(G), poly(U), poly(A), and double-stranded ribonucleotides] were from Pharmacia (Piscataway, NJ). Yeast RNA was purchased from Sigma (St. Louis, MO), and recombinant placental RNase inhibitor was from Gibco BRL (Grand Island, NY). Chemicals and some reagents, such as uranyl acetate dihydrate, perchloric acid, and polyacrylamide, were obtained from Fluka Chemical (Milwaukee, WI) or Bio-Rad Laboratories (Hercules, CA). Glassware, electrophoresis, and other apparatus for RNA assays were treated with RNase Erase (ICN Biomedicals, Aurora, OH) following recommendations of the manufacturer. Zymography. Zna2gp purified from human prostate (11) and epidermis (12) was separated on 12.5% SDS–PAGE gels (20 mg/lane). The RNase activities were assayed by zymography, with slight modification of a published protocol (18). Briefly, human epidermal total RNA, together with 2 mM EDTA, was added to the SDS–PAGE separating gel. After electrophoresis, the gel was washed twice for 20 min in 10 mM Tris–HCl, pH 8.0, also containing 20% isopropanol, under gentle shaking. This was followed by two 20-min washes in 10 mM Tris–HCl, pH 8.0, all at room temperature. The gel was then incubated in a buffer of 100 mM Tris–HCl, pH 8.0, and stained for 5 min with 0.5 mg/ml ethidium bromide. The bands were visualized under UV light and photographed. Dose response. Total RNA was extracted from the skin tissue of a healthy human donor (19). Twelve micrograms of this RNA was incubated at 37°C for 2 h with varying concentrations of prostate Zna2gp (from 12.5 to 500 ng), and the mixtures were denatured at 65°C for 5 min. They were then size fractionated on a 1.5% agarose–3% formaldehyde gel and then stained with 10 mM ethidium bromide. The resulting fluorescence intensity of the 28S and 18S rRNA bands was photographed under UV light. RNase assay. RNase activity, using yeast RNA as a substrate, was measured by the formation of perchloric acid soluble nucleotides, following a published procedure (20) with minor modification. Fifteen microliters of the substrates (5 mg/ml) was incubated with the enzyme in 250 ml of a buffer of 40 mM Tris–HCl, pH 7.5, containing 0.1 M NaCl and 5 mM EDTA. The reaction was carried out at 37°C for 30 min and then terminated by adding 50 ml of 25% perchloric acid containing 0.75% uranyl acetate. After further chilling at 4°C for 20 min, the reaction mixture was centrifuged at 13,600g for 15 min, 100 ml of supernatant was diluted with 1 ml of distilled water, and the absorbance was measured at 260 nm. RNase inhibition assay. Dose–response assays of RNase inhibition were performed with yeast RNA (8%) as the substrate (21). The reaction mixture contained 5 ml of various dilutions of a cloned placental RNase inhibitor and 25 ml of a reaction mixture consisting of 0.1 M Tris–HCl, pH 7.5, 10 mM EDTA, and 5 mM dithiothreitol, to which 5 ml of Zna2gp (0.6 ng) was added. RNase activity was measured after incubation at 37°C for 30 min. Alternatively, differ-

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FIG. 1. Identification and RNase activity of Zna2gp extracted from (A) prostate and (B) epidermis and of (C) recombinant epidermal Zna2gp. Lane 1, Coomassie-stained gels; the prostate Zna2gp migrated at 38 kDa, while that from the epidermis was at 34 kDa. Lane 2, RNase activities by zymography. Lane 3, corresponding Western blots.

ent concentrations of ZnCl2 or MgCl2 (up to 0.7 mM) were used as the putative inhibitor. Substrate specificity of Zna2gp RNase activity. RNase activity was assessed on single- and double-stranded synthetic polyribonucleotides by the formation of perchloric acid soluble nucleotides (22). Western blots. A duplicate SDS–PAGE gel of each Zna2gp was electrophoretically transferred to a nitrocellulose membrane in a buffer of 20% methanol, 25 mM Tris–HCl, and 192 mM glycine. The blot was quenched in TBST (10 mM Tris–HCl, pH 8.8, 150 mM NaCl, 0.05% Tween 20), containing 3% nonfat dried milk, for 30 min and then washed twice for 10 min in the buffer alone. The blot was incubated for 1 h with a 1:1500 dilution of rabbit antibody to human prostate Zna2gp in 0.5% nonfat dry milk in TBST and then washed twice for 10 min with unsupplemented TBST. It was then incubated for 1 h in a 1:3000 dilution of peroxidase-conjugated sheep antirabbit IgG (ECL Western blotting protocol, Amersham).

RESULTS

Identification of RNase Activity of Zna2gp Figure 1 depicts SDS–PAGE of prostate and epidermal Zna2gp, Western blots for each (using an antibody to prostate Zna2gp), and RNA zymograms of Zna2gp derived from both tissues. The prostate Zna2gp migrated at about 38 kDa, while that from the epidermis was at about 34 kDa. RNA was degraded by Zna2gp in the zymogels at the molecular mass corresponding to the bands in SDS–PAGE and in the Western blots; the enzymatic activity appeared to be comparable for prostate and epidermal Zna2gp. As a control (to rule out a contaminant), recombinant epidermal Zna2gp was also used; it gave results similar to those for native epidermal Zna2gp. Using a formaldehyde–agarose gel assay, the enzymatic activity on epidermal RNA was observed in dose–response fashion in the range 25–500 ng of prostate Zna2gp (Fig. 2). In view of the reported substantial homology of Zna2gp with MHC class I, we also tested six HLA class I heavy chains (HLA-A2, HLA-B8, HLA-CW3, HLACW6, HLA-CW7, and HLA-G). We could not detect RNase activity on zymogels with any of them (data not shown).

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FIG. 2. Dose response for RNase activity of Zna2gp. Lane 1 was buffer control. Lanes 2–7 contained 12.5, 50, 100, 200, and 500 ng of Zna2gp, respectively.

Dependence of RNase Activity of Zna2gp on pH, Ionic Strength, and Temperature We used yeast RNA (8%) as substrate to characterize the RNase activity of Zna2gp (the activity on yeast RNA was 85% of that on human epidermal total RNA). As a function of pH, it was strongest in the 6 – 8 range, with a peak at 7.5 (Fig. 3). The influence of ionic strength was weaker (Fig. 4), with a maximum at an NaCl concentration of 50 mM. The temperature variation had comparable variation over the range from 20 to 80°C, with a maximum at 60°C (Fig. 5).

FIG. 4. Relative RNase activity of Zna2gp as a function of ionic strength (concentration of NaCl).

activity of Zna2gp was markedly inhibited by very low concentrations of ZnCl2 in dose–response fashion, almost halved at 0.1 mM; it remained unaffected by MgCl2. Action of Zna2gp on Single- and Double-Stranded Synthetic Polyribonucleotides The RNase activity of prostate Zna2gp was assayed spectrophotometrically under identical experimental conditions on various single- and double-stranded polyribonucleotides. Zna2gp showed a clear preference for pyrimidine [poly(C) and poly(U)] and cleaved only single-stranded RNA (Fig. 8).

Inhibition of RNase Activity of Zna2gp We measured the effect of cloned placental RNase inhibitor on the enzyme activity of Zna2gp with a yeast RNA substrate. Figure 6 shows that the RNase activity decreased to half at an inhibitor concentration of 1.25 U for samples containing 0.6 ng of Zna2gp (U is defined as the amount required to inhibit 5 ng of RNase A by 50%) but only dropped asymptotically to about 20%. The effect of the cations Zn21 and Mg21 is shown in Fig. 7: the RNase

DISCUSSION

FIG. 3. Relative RNase activity of Zna2gp on yeast RNA as a function of pH.

FIG. 5. Relative RNase activity of Zna2gp as a function of temperature (°C).

To put the ribonuclease activity of Zna2gp in context, we compared it with two prominent RNases: human pancreatic RNase A and frog oocyte onconase. The three ribonucleases show a number of similarities, but also exhibit structural and catalytic differences. Most obviously, they differ in size: RNase A and onconase have molecular weights in the range of 12–14 kDa,

ZINC-a2-GLYCOPROTEIN HAS RIBONUCLEASE ACTIVITY

FIG. 6. Inhibition of RNase activity of Zna2gp by cloned placental RNase inhibitor (dose response). The inhibitor unit, U, is defined as the amount required to inhibit 5 ng of RNase A by 50%; the samples contained 0.6 ng of Zna2gp.

while Zna2gp is at 35– 40 kDa (depending on posttranslational modifications). The activities of the ribonucleases are substrate dependent. All three degrade ribosomal RNA more strongly than yeast RNA. They preferentially cleave ssRNA substrates. It has been speculated that such RNases play a role in preventing invasion by prokaryotic or eukaryotic organisms, while other enzymes, which degrade dsRNAs, are involved in antiviral action (22). All three are pyrimidine specific, but RNase A shows a strong preference for poly(C) and much less for poly(U) (22), onconase prefers poly(U) (23), and Zna2gp responds equally to poly(C) and poly(U). The specific activity of Zna2gp is similar to that of onconase and is two orders of magnitude lower than that of RNase A. The three ribonucleases have similar optima in pH [at 8.0 for RNase A and at 6.0 for onconase (24), at 7.5 for Zna2gp] and in temperature (at about 60°C) on yeast RNA. Their sensitivity to the ionic strength of the reaction mixture has a broad maximum: onconase activity peaks at 20 mM NaCl, bovine RNase A at 40 mM and human RNase A at 100 mM (23), and Zna2gp at 50 mM. While Zn21 ions inhibit the activities of all three RNases, Mg21 ions do not. RNase A is extremely sensitive to placental ribonuclease inhibitor, while onconase is unaffected. The insensitivity of onconase to inhibitors may contribute to its antiproliferative and cytotoxic properties (25, 26). Zna2gp is partially inhibited (down to 20%). We have preliminary data indicating that Zna2gp inhibits the proliferation of a tumor cell line. RNase A cannot compete with onconase in the induction of its cellular toxicity, suggesting that the two enzymes bind to different cell-surface receptors (25). The cell-surface receptor of Zna2gp remains undetermined. The N-terminus of onconase is pyroglutamic acid (pyr 1), and it is in the region of the active site. Its replacement by another amino acid residue drastically reduces enzymatic activity; when pyr 1 is reconsti-

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FIG. 7. Effect of divalent cations on RNase activity of Zna2gp (relative activity vs concentration of MgCl2 or ZnCl2).

tuted, onconase regains its enzyme activity (23). Zna2gp has the same pyr 1 N-terminus. In contrast, the N-terminus of RNase A is a lysine, and it occurs outside the active site; substitution of the N-terminal lysine with another amino acid has no effect on the RNase activity (23). Because of repeated reference in the literature to the 36 –39% homology of Zna2gp to MHC class I antigens, we tested several HLA heavy chains for RNase activity and found none; incidentally, MHC class I heavy chains do not contain a pyr 1 at their N-terminus. Zna2gp has fewer commonalities with onconase or RNase A. Alignment of the amino acid sequences of Zna2gp and onconase, using the Gene Runner program, reveals scattered homologous segments (Table I). Matches between Zna2gp and RNase A are even more fragmentary (not illustrated). Apparently, the extent of overall structural homology (at a moderate level) is not a good indicator of function, whatever its evolutionary implications. Conversely, appreciable interspecies structural differences have been noted within the ribonuclease superfamily, such as between human and bovine RNase A (27). The RNase activity reported here may be the key to the physiological function of Zna2gp. Ribonucleases are widely distributed in various organs and body fluids, where their

FIG. 8. Relative RNase activity of Zna2gp on synthetic polyribonucleotides, both single stranded (A, C, G, U) and double stranded (A:U, C:G). Note logarithmic scale.

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LEI ET AL. TABLE I

Alignment of Amino Acid Sequences of Zna2gp and Onconasea Zna2gp Onconase

1 QE QE 1

11 TYIY TFIY 36

18 SKHVEDVPAF SKNVLTTSEF 55

88 Y Y 65

166 CPATLRKYLKYSK CNVTSRPCKYKLK 69

190 VVTSHQAP VTCRNQAP 89

217 VHWTAAG VHFVGVG 97

a

Overall alignment obtained with Gene Runner. In view of the limited homology, only segments in which matches occur are shown, with breaks indicating omissions. Sequence numbers are identified at the beginning of each block.

biological activity has been associated with infection, immune regulation, and antitumor activity (24). As a soluble molecule, Zna2gp is likely to function both intra- and extracellularly to degrade and eliminate by-products of RNA processing reactions within cells, as well as incorrectly synthesized or damaged RNAs (including some that compete with functional RNAs). Degraded RNAs may supply cell nutrients for the continual readjustment of the mRNA pools to the changing needs of the cell (28).

12.

13.

14. 15.

ACKNOWLEDGMENTS We are grateful for gifts of prostate Zna2gp from H. Haupt, of antibody to it from I. Ohkubo, and of the HLA molecules from J. Strominger.

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