©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Active Carbonyl Formed during Glycosylphosphatidylinositol Addition to a Protein Is Evidence of Catalysis by a Transamidase (*)

(Received for publication, May 22, 1995; and in revised form, June 19, 1995 )

Stephen E. Maxwell Sandhya Ramalingam Louise D. Gerber Larry Brink Sidney Udenfriend (§)

From the Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110-1199

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumably the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.


INTRODUCTION

Proteins that are anchored to membranes via a glycosylphosphatidylinositol (GPI) (^1)linkage, although discovered relatively recently(1, 2) , are now known to be ubiquitous among eukaryotes where they serve a variety of important functions(3, 4, 5, 6, 7, 8, 9) . Some GPI anchored proteins are known to be required for the survival of yeast (10) and of human erythrocytes(11, 22) . Much is already known about the structure and pathway of biosynthesis of the GPI moiety(13, 14, 15, 16, 17) . A fair amount has also been learned about the structural requirements of a protein in order for it to condense with GPI(18, 19, 20, 21, 22, 23) . However, the mechanism of the condensation and the enzyme(s) that catalyzes it have yet to be elucidated.

A GPI-anchored protein is coded for as a preproprotein containing both NH(2)- and COOH-terminal signal peptides (Fig. 1). As the nascent chain is translocated into the endoplasmic reticulum the NH(2)-terminal signal peptide is removed giving rise to the proprotein. Cleavage of the COOH-terminal signal peptide from the proprotein occurs concomitantly with the condensation of the ethanolamine group of GPI with the COOH group of the newly formed COOH-terminal amino acid ( site). Apparently both processes occur in a single step catalyzed by what has been assumed to be a transamidase. We have studied this process using rough microsomal membranes (RM) from various cells coupled to a translational system with mRNA coding for an engineered form of human placental alkaline phosphatase (PLAP) as a template. In the latter, the catalytic domain and all the glycosylation sites were removed to form a much smaller product (miniPLAP). With this system, the translation of preprominiPLAP mRNA and the subsequent steps in its processing to mature GPI-linked miniPLAP can be readily monitored (Fig. 1) (24, 25) (^2)Processing of GPI proteins by a transamidase would involve a nucleophilic attack by the ethanolamine group of GPI on an activated carbonyl on the residue of the pro form of a protein. In a previous report(26) , we presented evidence that water, a highly abundant nucleophile, competes with GPI and gives rise to a mature form of miniPLAP (free miniPLAP) that is also cleaved at the site but does not contain the GPI anchor. In RM from most cells, although GPI-linked miniPLAP is the major product, it is always accompanied by some free miniPLAP (Fig. 1). In RM from GPI-deficient cells, free miniPLAP is formed in amounts comparable to the GPI form(26) . Hydrazine and hydroxylamine are known to be nucleophilic acceptors in transpeptidase (27) and transamidase (28) reactions. To obtain additional evidence that processing to GPI proteins involves a transamidase, we investigated hydrazine and hydroxylamine as possible substrates in our cell-free system.


Figure 1: Schematic presentation of processing of a proprotein to a GPI protein by RM. The NH(2)-terminal signal peptide is cleaved once the protein has been introduced into the ER. Any preproprotein that is isolated with RM is protease sensitive and does not exist in the context of the membrane. The proprotein apparently requires energy, the chaperonin BiP and GTP for proper folding and/or translocation to the site of the transamidase. The latter presumably combines with the proprotein through the COOH group of the site amino acid to form an activated carbonyl group. Nucleophilic attack by the ethanolamine component of GPI on the carbonyl group combines GPI with the protein in amide linkage to yield the GPI-protein with concomitant cleavage of the COOH-terminal signal peptide. Water is also capable of attacking the site carbonyl group to yield the free protein with concomitant release of the COOH-terminal signal peptide. The inset shows a typical gel with the products (after immunoprecipitation and SDS-PAGE) obtained by cotranslational processing of miniPLAP mRNA in the presence of RM from cells that produce GPI.




EXPERIMENTAL PROCEDURES

Reagents

Radiolabeled compounds were from Amersham Corp. Unless otherwise stated, all chemical reagents were from standard commercial sources. Stock solutions (1.0 M) of hydrazine dihydrochloride and hydroxylamine monohydrochloride were prepared in water and adjusted to pH 7.0 with NaOH. Human PLAP (about 1% purity) was obtained from Sigma. Phosphatidylinositol-specific phospholipase C (PI-PLC) was purified as described by Taguchi et al.(29) from Bacillus thuringiensis. Variant surface glycoprotein (VSG), labeled with [^3H]myristic acid, isolated from Trypanosoma brucei MIT 118A was a generous gift of G. A. M. Cross of Rockefeller University. Affinity purified antiPLAP IgG was from Accurate Chemical and Scientific Corp (Westbury, NY).

Cell Culture and Preparation of Rough Microsomal Membranes

All media contained 10% heat-inactivated fetal bovine serum. HeLa cells were grown in a Biolafitte bioreactor in Iscove's modified Dulbecco's medium at 37 °C. Several different GPI-deficient cell lines were used. The Ltk cell line, a derivative of L929 cells which is deficient in synthesizing the GPI intermediate N-acetylglucosamine phosphatidylinositol (30) , was grown in Dulbecco's modified Eagle's medium. The C1 cells, a derivative of M31/25 cells were grown in RPMI 1640 supplemented with 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The M31/25-C1 cells are a derivative of YH16/33 cells and are deficient in transferring N-acetylglucosamine to phosphatidylinositol(31) . Both Ltk and M31/25 cells were gifts from E. T. H. Yeh of Texas Biotechnology Corporation. RM were prepared as described previously (26) and resuspended in 250 mM sucrose, 50 mM triethanolamine (TEA), pH 7.5, to a final concentration of 50 A units/ml. These preparations of RM were frozen on dry ice and stored at -70 °C.

Cotranslational Processing of MiniPLAP

Unless otherwise noted, translations were carried out with mRNA encoding preprominiPLAP Ser-208 which was transcribed from the corresponding cDNA (37) and purified using an RNaid kit (Bio 101). Translations were carried out in 25-µl volumes at 30 °C using a rabbit reticulocyte lysate (Promega) as described previously (32) except that the 2 min preincubation prior to addition of RM was omitted. Times of incubation are in the figure legends. The RM from each cell type were added to a final concentration of 8 A units/ml. Where indicated, 1.0 µl of 260 mM hydrazine or hydroxylamine was added to yield a final concentration of 10 mM. Maximal rates of formation of 23-kDa products were achieved at somewhat lower concentrations of the two reagents, but 10 mM was chosen to ensure saturation.

Preparation of RM Preloaded with prominiPLAP

When cotranslational processing is carried out for a short period of time isolated RM contain large amounts of prominiPLAP, the substrate of the putative transamidase, and only small amounts of GPI linked miniPLAP (33) . Such RM are considered to be preloaded since on further incubation under suitable conditions prominiPLAP is converted to GPI-linked miniPLAP. Scaled up preparations of preloaded RM were made essentially as described previously(33) . Prior to addition of RM the translation mixture (84.0 µl) was incubated for 2 min at 30 °C. The RM (16.0 µl) were then added, and the mixture was incubated for an additional 18 min at 30 °C and then placed on ice for 5 min to stop the reaction. Samples were pooled and 200 µl of reaction mixture were layered over a step gradient consisting of a 100-µl spacer layer (250 mM sucrose, 50 mM TEA, pH 7.5) over a 500-µl cushion of 500 mM sucrose, 50 mM TEA, pH 7.5. Following centrifugation at 267,000 g for 15 min at 4 °C in a TLA 100.2 rotor, the supernatants were removed and the pellets rinsed with 500 µl of the 250 mM sucrose, 50 mM TEA, pH 7.5, buffer. The same buffer was used to resuspend the preloaded RM (200 µl of buffer/600 µl of starting material). RM preloaded for 60 min were prepared in the same manner except that after pelleting they were resuspended in half of their original volume. Under these conditions the GPI-linked form is the major miniPLAP product.

Post-translational Processing of Preloaded RM

Preloaded RM (12.5 µl) were diluted with an equal volume of buffer containing 20 mM creatine phosphate, 100 µg/ml creatine phosphate kinase, 4 mM dithiothreitol, 160 mM potassium acetate, 8 mM magnesium acetate, 1 mM ATP, 1 mM GTP, and protease inhibitors (aprotinin, antipain, leupeptin, chymostatin, and bestatin) 4 µg/ml each. Hydrazine, hydroxylamine, or water were added (1 µl) prior to incubation at 30 °C for the time indicated.

Treatment of Isolated PLAP or VSG with Either PI-PLC, Hydrazine, or Hydroxylamine

PI-PLC cleaves the GPI anchor between the phosphate group of phosphatidylinositol and the hydrophobic diacylglycerol moiety. PI-PLC treatment of PLAP was carried out in a final volume of 40 µl containing 150 pmol of PLAP, 50 mM Tris-HCl, pH 7.5, and 200 units of PI-PLC (34) . For [^3H]myristyl VSG, 0.5 µg of protein (5000 counts/min) was treated with 17 units of PI-PLC in a final volume of 25 µl of the same buffer. Samples were incubated for 2.5 h at 30 °C. Treatment with hydrazine or hydroxylamine (100 mM) was carried out under the same conditions.

Phase Partitioning of PLAP

Because of its GPI moiety PLAP is a fairly hydrophobic protein and under certain conditions much of it can be extracted from an aqueous into an organic phase. To determine whether GPI had been cleaved by any of the procedures that were used, phase separations were performed as described by Bordier(35) . Triton X-114 was the organic phase and 0.15 M NaCl the aqueous phase. Following separation of the two phases, the aqueous layer was removed, and an aliquot was immediately assayed for PLAP enzyme activity using the method of McComb and Bowers(36) .

Butanol Extraction of Lipids Derived from VSG

Any lipid material cleaved from [^3H]myristyl VSG following various treatments in 25 µl was extracted into 500 µl of water-saturated butanol by vortexing vigorously for 1 min. The samples were then centrifuged at 14,000 g for 2.5 min, and a 400-µl aliquot of the butanol was removed for measurement of radioactivity in a liquid scintillation counter.

Immunoprecipitation and SDS-PAGE

Samples were diluted with an equal volume of 10% SDS, 8% beta-mercaptoethanol (v/v) and boiled for 5 min. After cooling to room temperature, an equal volume of water was added, and aliquots were removed for immunoprecipitation with affinity purified rabbit polyclonal IgG (38) followed by SDS-PAGE(26) . The gels were fixed in 10% acetic acid containing 45% methanol, dried, and exposed to a phosphorimaging screen. Results were visualized and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Background radiation was subtracted using the object average function of ImageQuant version 4.1 (Molecular Dynamics). For calibration purposes, [S]methionine-labeled miniPLAP-Delta179 (free miniPLAP) was expressed using the corresponding mRNA (21) in the cotranslational system. Aliquots of immunoprecipitated protein containing increasing amounts of radioactivity were subjected to SDS-PAGE, then monitored, and quantified on the PhosphorImager. Linearity was shown over the entire range of density obtained in all experiments. Data in the figures are based on duplicate samples from a single experiment. In each case, similar values were obtained in two or more independent experiments carried out on different days and with different preparations of RM.


RESULTS

We have used two types of experimental conditions to study GPI addition in cell-free systems, cotranslational and post-translational. In the latter the translational elements are separated from the RM after a defined period of translation(33) . We refer to these as preloaded RM. During short periods of incubation, RM from HeLa cells can be preloaded so that they contain an appreciable amount of substrate, prominiPLAP, but little mature GPI-linked or free miniPLAP. The post-translational system was chosen for initial studies because we felt that it provided a more direct study of COOH-terminal processing and were concerned that hydrazine and hydroxylamine might interfere with translation in our cotranslational system. What we were looking for was conversion of prominiPLAP to either the hydrazide or hydroxamate of miniPLAP. Since the latter substituents are fairly small (31 and 32 Da, respectively), the miniPLAP derivatives would be expected to migrate with free mature miniPLAP on SDS-PAGE.

Initial studies were carried out with HeLa RM preloaded for 20 min. Surprisingly, when these were incubated further, the amount of GPI-linked miniPLAP (24.7 kDa) initially present in the preloaded RM was reduced accompanied by an even larger increase in 23-kDa product(s) (data not shown). This unexpected finding could be explained by an exchange in which the GPI anchor was removed from miniPLAP and the corresponding hydrazide or hydroxamate formed. The following experiments were carried out to verify this.

Conversion of GPI-linked miniPLAP (24.7 kDa) in Preloaded RM to a 23-kDa Product in the Presence of Hydrazine or Hydroxylamine

The unexpected observation that both hydrazine and hydroxylamine could apparently cleave the GPI moiety from mature GPI-linked miniPLAP in preloaded RM was further investigated using HeLa RM that were preloaded for 60 min rather than the usual 20 min. The longer than usual preloading time was to increase the amount of mature GPI-linked protein prior to addition of the nucleophilic agents. Under those conditions, about 60% of the total amount of all forms of miniPLAP in the preloaded RM was present as the mature GPI-linked form (Fig. 2, lanes A and B). There was also a small amount of 23-kDa material as free miniPLAP. On further incubation for 30 min, there was no additional processing (Fig. 2, lanes C and D). However, when incubations were carried out in the presence of hydrazine or hydroxylamine for the additional 30 min the amount of GPI-linked miniPLAP decreased by almost 50% accompanied by an increase in 23-kDa product(s), presumably representing the corresponding hydrazide or hydroxamate of miniPLAP (Fig. 2, lanes E and G). When the RM were heated prior to addition of the nucleophilic reagents conversion to a 23-kDa product was markedly diminished (Fig. 2, lanes F and H), strongly suggesting that the reaction was enzyme catalyzed. The following experiments were carried out to rule out the possibility of chemical cleavage of GPI from the protein by the two nucleophilic agents.


Figure 2: Processing of prominiPLAP in preloaded HeLa RM. The RM were preloaded by incubating them in the presence of miniPLAP mRNA and the translational system for 60 min, following which they were isolated by differential centrifugation. Preloaded RM were then incubated for an additional 30 min then immunoprecipitated and subjected to SDS-PAGE as described under ``Experimental Procedures.'' Heated samples were incubated at 50 °C for 15 min prior to the 30 min incubation. Toppanel, images of the gels in the PhosphorImager. Bottom panel, the amount of each protein species was quantified on the PhosphorImager, and the values on the y axis represent relative density units. Preload indicates the composition at the end of the preloading period with no additional incubation. Control RM were incubated for 30 min in the presence of buffer alone; HDZ represents incubation in the presence of 10 mM hydrazine and HXL, incubation in the presence of 10 mM hydroxylamine. One of two duplicate gels are shown (top panel). The relative densities (bottom panel) represent the average (±10%) of the values obtained in each of the duplicate gels.



Does chemical cleavage of GPI from proteins occur in the presence of hydrazine or hydroxylamine

To demonstrate further that the cleavage of GPI-linked proteins by hydrazine and hydroxylamine is catalyzed by an enzyme resident in functional RM and not simply through chemical cleavage, we treated two isolated GPI-linked proteins, PLAP and VSG with either hydrazine, hydroxylamine, or PI-PLC. The latter, which is known to cleave diacylglycerol from the GPI moiety(14) , served as a positive control. Following each treatment, reaction mixtures were subjected to partitioning between Triton X-114 and 0.015 M NaCl to detect cleavage of a hydrophobic moiety. In the case of untreated PLAP, 20% of the enzyme activity appeared in the aqueous phase after partitioning between detergent and the aqueous phase (Fig. 3). This is in accord with previous reports(32, 39) . Removal of the lipid moiety of GPI from PLAP by treatment with PI-PLC reversed the pattern of distribution, 80% now remaining in the aqueous phase. However, when PLAP was treated with either hydrazine or hydroxylamine there was no change in its distribution (20% remaining in the aqueous phase) indicating that the GPI anchor had not been cleaved from the protein by either of the nucleophilic agents.


Figure 3: Effect of PI-PLC, hydrazine, or hydroxylamine on PLAP. Following treatment with each reagent, aliquots of the reaction mixtures were subjected to partitioning between 0.15 M NaCl and Triton X-114, and PLAP enzyme activity was measured in the aqueous phases. Each sample contained 20 units of PLAP activity. HDZ represents incubation in the presence of 100 mM hydrazine and HXL, incubation in the presence of 100 mM hydroxylamine.



To investigate the effects of hydrazine and hydroxylamine on another GPI protein, VSG, the glycolipoprotein, labeled with tritium in its myristic acid residues, was treated with either of the two nucleophiles or with PI-PLC. It is known that diacylglycerol, produced by treatment of VSG with PI-PLC and free GPI that may be formed, are extractable into butanol, whereas VSG is not(40) . As shown in Fig. 4only 3% of the radioactivity of untreated VSG was extracted. After PI-PLC treatment 97% of the radioactivity was extracted by butanol. In contrast, on treatment of VSG with hydrazine or hydroxylamine the radioactivity extractable by butanol was again only about 3%. As with PLAP, the nucleophilic agents, by themselves, did not cleave the GPI moiety from VSG. It should be noted that concentrations of the nucleophiles at least 10 times higher were used here than in the studies with RM. These findings are further evidence that the conversion of GPI miniPLAP (24.7 kDa) to a 23-kDa moiety in RM in the presence of 10 mM hydrazine or hydroxylamine at neutral pH and 30 °C is not due to chemical cleavage but is enzymatically catalyzed.


Figure 4: Effect of PI-PLC, hydrazine, and hydroxylamine on VSG containing [^3H]myristic acid. Total radioactivity was 5000 counts/min/sample. Following reaction, each sample was extracted with n-butanol and an aliquot taken for measurement of radioactivity. HDZ represents incubation in the presence of 100 mM hydrazine and HXL, incubation in the presence of 100 mM hydroxylamine.



Our original goal was to demonstrate an interaction of prominiPLAP with the nucleophilic agents that would yield a product of essentially the same mass as free mature miniPLAP. However, in RM from HeLa cells the relatively large amounts of free miniPLAP formed under preloading conditions (Fig. 2) and the contribution of 23-kDa material produced by the enzyme-catalyzed cleavage of GPI-linked miniPLAP by the nucleophilic agents, presumably the corresponding hydrazide or hydroxamate of miniPLAP, obscured any contribution of 23-kDa product(s) that might arise from a direct interaction of the nucleophiles with prominiPLAP. Nevertheless, we consistently observed 30-40% increases in the total amount of mature miniPLAP species (24.7 kDa plus 23 kDa) in the presence of the nucleophiles, all in the 23-kDa product(s) suggesting that some of the 23-kDa material arose by direct interaction of the nucleophilic agents with prominiPLAP (27 kDa). Cotranslational experiments with RM from various cells were carried out to explore this further.

Cotranslational Processing in the Presence of Hydrazine

For the reasons stated above, experiments with preloaded RM from HeLa cells only suggested that both hydrazine and hydroxylamine could react with an activated transamidase-prominiPLAP intermediate to cleave the signal peptide and yield the corresponding hydrazide or hydroxamate of miniPLAP. In spite of the possibility that the nucleophilic agents would interfere with translation, we elected to test them in the cotranslational system. Hydroxylamine did interfere with translation and studies with it were abandoned. However, hydrazine up to 10 mM was without effect on translation. All further experiments were, therefore, carried out in the presence of 10 mM hydrazine. It should be noted that this concentration is only one-tenth of that used in the treatment of isolated PLAP or VSG in the absence of RM.

Cotranslational processing by RM from HeLa cells in the presence of hydrazine caused a reproducible (30%) increase in the total amount of the mature forms of miniPLAP, (24.7 and 23 kDa) (Fig. 5, lanes A and B). Again such an increase (all in the 23 kDa species) is consistent with the formation of some miniPLAP hydrazide directly from prominiPLAP. We therefore turned to RM from GPI-deficient Ltk and M31/25-C1 cells because of their limited ability to produce GPI-linked proteins while producing large amounts of the precursor prominiPLAP. Such RM also produce much less free mature miniPLAP for reasons stated previously (26) . If hydrazine could act as an effective acceptor for an enzyme activated carbonyl intermediate, its addition to the cotranslational system should increase the amount of 23-kDa product(s) processed by the RM. As shown in Fig. 5, cotranslational processing of miniPLAP mRNA in the presence of hydrazine resulted in a 200% increase in the total amount of mature products in Ltk RM (lanes C and D) and a 110% increase in C1 RM (lanes E and F). Most importantly, in each case, all of the increase was in the 23-kDa product(s), consistent with the formation of the hydrazide of miniPLAP.


Figure 5: Cotranslational processing of mRNAs of Ser and Gly miniPLAP in the presence of hydrazine. Following incubation in the presence of 10 mM hydrazine, samples were treated and assayed as described in the legend to Fig. 2. Both gels were exposed on the same PhosphorImager screen simultaneously. Because Ser miniPLAP is a much better substrate than Gly miniPLAP, the PhosphorImager contrast was increased for the latter, hence the more intense image. This instrumental manipulation does not affect quantitation. The wide range of densities made it necessary to expand the scale to present the data more clearly to the reader. Group I represents the full scale between 0-400 10^4 relative density units; group II expands the range on scale I from 0 to 100 10^4; group III, expands the scale from 0 to 20 10^4. It should be noted that the relative density of the 23 kDa band in lane L, shown in group III is about 10 background. As in Fig. 2, one of two duplictae gels is shown (top panel) and relative densities are the average (±10%) of the duplicates (bottom panel). HDZ represents incubation in the presence of 10 mM hydrazine.



The studies above were all carried out with Ser miniPLAP mRNA. While prominiPLAP with serine at the site is by far the best precursor for GPI addition(41) , it also yields the largest amount of free mature miniPLAP, presumably because the putative carbonyl form of serine reacts to an inordinate extent with water compared to other amino acids at that site(26) . To minimize the amount of free mature miniPLAP formed due to hydrolysis of an intermediate, we utilized Gly miniPLAP mRNA. As shown in Fig. 5(lane G), the proportion of prominiPLAP converted to mature GPI-linked miniPLAP (24.7 kDa) in HeLa RM was markedly reduced and, as we expected(26) , smaller amounts of the hydrolysis product (23 kDa) appeared. Addition of hydrazine (lane H) during cotranslational processing by HeLa RM increased the amount of 23-kDa product(s) with a small increase in total products (24.7 and 23 kDa). Again we found it necessary to use RM from the mutant cells Ltk and M31/25-C1 to minimize the production of GPI-linked proteins. The findings clearly indicate that in the presence of hydrazine, processing to mature products is increased; 114% for Ltk RM (Fig. 5, lanes I and J) and 44% for RM from M31/25-C1 cells (lanes K and L). Furthermore, in each case all the increase was in a product that migrated with a mass of 23 kDa. This is again consistent with the formation of the hydrazide of mature miniPLAP concomitant with cleavage of the signal peptide. Observations similar to the ones with glycine were also obtained with constructs containing aspartic acid, alanine, and cysteine at their sites (data not shown).

Studies with Preloaded RM from Ltk Cells

All the above experiments show convincingly that both hydrazine and hydroxylamine catalyze the formation of 23-kDa product(s) in amounts greatly in excess of the GPI-linked miniPLAP formed in the absence of the nucleophiles. However, they do not entirely rule out the possibility of GPI-linked miniPLAP being an obligatory intermediate in the formation of 23-kDa product(s). To investigate this further, studies with preloaded RM from GPI-deficient Ltk cells were carried out. As shown in Fig. 6(lane A), the preloaded RM contained small amounts of GPI-linked miniPLAP and a somewhat larger amount of 23-kDa product(s). On further incubation of the preloaded RM, there was a gradual increase in free miniPLAP (23 kDa) reaching maximal values by 60 min (lane B). When incubation was carried out in the presence of the nucleophilic agents there were also gradual increases in 23-kDa product(s) with essentially no change in the amount of GPI-linked miniPLAP. Again maximal values were attained by about 60 min (lanes D and F). Heated RM were totally inactive. In fact, with hydrazine and hydroxylamine the amounts of 23-kDa product(s) formed were far greater than the total of 24.7- and 23-kDa products originally present in the preloaded RM. This could only have arisen by direct conversion from prominiPLAP.


Figure 6: Processing of prominiPLAP in preloaded RM from GPI-deficient cells. RM from Ltk cells were preloaded for 20 min. After an additional incubation of 60 min, samples were treated and assayed as described under the legend to Fig. 2. Data are presented as in the legend to Fig. 2. HDZ represents incubation in the presence of 10 mM hydrazine and HXL, incubation in the presence of 10 mM hydroxylamine.




DISCUSSION

While the overall biosynthetic pathway of GPI proteins has been deduced and has been demonstrated in cell-free systems, one of the key components of the overall reaction, the enzyme(s) that catalyzes condensation of the ethanolamine of GPI with the COOH at the site of a proprotein along with the elimination of a signal peptide remains elusive. In fact, evidence that the condensing enzyme is a transamidase is still only indirect. Ferguson and Williams (14) concluded that the reaction is so rapid that condensation and signal peptide elimination must be concomitant reactions as would be catalyzed by a transamidase. Mayor et al.(42) have shown that membrane fractions from trypanosomes incorporate GPI into VSG (i.e. condense the ethanolamine of GPI with the carboxyl of pro-VSG) in the absence of an energy source. Production of an amide or a peptide bond requires energy unless it is catalyzed by a transamidase or transpeptidase. Amthauer et al.(33) showed that while ATP increased conversion of prominiPLAP to GPI-linked miniPLAP in RM, the energy was required to release the chaperonin BiP and not for formation of the amide bond of the GPI protein. More recently, Maxwell et al.(26) demonstrated that during the cell-free processing of prominiPLAP in RM from GPI-deficient cells, while the major product was GPI linked, as much as 40% was cleaved at the correct site, but with the addition of the elements of water instead of GPI. Evidence was presented that the latter product arose through a competition of water, an abundant nucleophile, with the ethanolamine of GPI, for condensation with an active carbonyl moiety at the site of prominiPLAP. Activation of a carboxyl group in the absence of an energy source so that it can condense with an appropriate nucleophile is the hallmark of a transamidase.

The present studies with hydrazine and hydroxylamine give further credence to the transamidase nature of the condensing enzyme. Nevertheless, these experiments, too, are not as conclusive as they could be because the amounts of product formed were too small (10-100 fmol) to permit chemical identification. There is a colorimetric assay for hydroxamates(43) , but it requires far greater amounts of material than were formed in these studies. Another factor that makes clear interpretation difficult is that the reaction products of the nucleophiles with prominiPLAP, the corresponding hydrazide or hydroxamate of miniPLAP, are of about the same size as free miniPLAP, 23 kDa. The latter, which is formed by the reaction of prominiPLAP with water, always appears as a side product during the processing of prominiPLAP. It would, therefore, be expected to comigrate with any hydrazide or hydroxamate of miniPLAP that was formed. At present we can only surmise that the 23-kDa products formed in the presence of the nucleophilic agents are the corresponding hydrazides or hydroxamates. On the positive side, hydrazine, in the cotranslational system, increased the amount of 23-kDa product(s) 2-3-fold when added to RM from GPI deficient Ltk and M31/25-C1 cells. These large increases did not occur when the RM were heated prior to treatment with hydrazine or when incubations were carried out at 4 °C. In addition, reaction with hydrazine or hydroxylamine was found to be concentration- and time-dependent. All of the above are clear indicators of enzyme catalysis. The findings with preloaded GPI-deficient RM (Fig. 6) are most cogent. They show that GPI-linked miniPLAP is not an intermediate in the conversion of prominiPLAP to 23-kDa product(s) in the presence of hydrazine and hydroxylamine. There remains the remote possibility that for some reason GPI-linked miniPLAP (24.7 kDa) is formed but does not accumulate in Ltk RM as it does in RM of cells that produce normal amounts of GPI.

The finding that both hydrazine and hydroxylamine can convert preformed GPI miniPLAP (24.7 kDa) to 23-kDa product(s) in the presence of RM was at first disturbing. However, this reaction too is enzyme catalyzed since heated or disaggregated RM were inactive. Furthermore, hydroxylamine and hydrazine, at concentrations far higher than were used in the experiments with RM, did not cleave GPI from two isolated GPI proteins, PLAP and VSG. In addition, GPI-linked miniPLAP, isolated from HeLa RM that had been detergent extracted and heated, was not cleaved by the nucleophilic agents (data not shown). The best explanation for a catalytic conversion of GPI-miniPLAP to the putative hydrazide or hydroxamate with the elimination of GPI is the following. In our in vitro system, at least, the GPI protein remains in contact with the transamidase. As in prominiPLAP, the carboxyl of GPI-miniPLAP could then be activated to a carbonyl moiety which in turn could be attacked by potent or more abundant nucleophiles such as hydrazine or hydroxylamine. In the process the GPI moiety would be cleaved and the nucleophile elements added.

There are precedents for such an enzymatically catalyzed attack of hydrazine or hydroxylamine on the end product of a transamidation reaction. For example, -glutamyl transpeptidase (GT) normally catalyzes the transfer of a glutamate residue of glutathione to cysteine to form -glutamyl-cysteine(27) . The latter, in the presence of GT and hydrazine or hydroxylamine can be converted to the -glutamyl hydrazide or hydroxamate with the release of cysteine. In this case, cysteine in the dipeptide may be considered to be the equivalent of GPI. Another example of a nucleophilic attack of hydrazine on an enzymatic product of a transpeptidase in the presence of the enzyme is provided by glutaminase(28) . In accord with the above, the enzyme-catalyzed exchange of hydrazine or hydroxylamine for GPI on mature miniPLAP does not require the addition of ATP or GTP to the preloaded RM and occurs even in the presence of nonhydrolyzable analogs of ATP or GTP (data not shown).

The nucleophiles unquestionably cleave the GPI moiety from miniPLAP and apparently form the corresponding hydrazide or hydroxamate. This raises the question whether the 23-kDa material that is formed in the presence of hydrazine or hydroxylamine arises by a direct nucleophilic attack on prominiPLAP as in Fig. 7(c and d), or in two steps with GPI miniPLAP as an intermediate (Fig. 7, a and e or a and f). The large increases in 23-kDa product(s) formed cotranslationally in RM, particularly from Ltk and M31/25-C1 cells, unquestionably indicate that the reaction with hydrazine by whichever pathway is enzyme catalyzed. Since even in the presence of hydrazine some GPI miniPLAP appears, it is likely that under cotranslational conditions miniPLAP hydrazide (23 kDa) arises via both pathways. However, the findings with preloaded RM from Ltk cells clearly show that 23-kDa product(s) can also be formed directly from prominiPLAP without GPI miniPLAP appearing as an intermediate (Fig. 7, c and d). Thus, it may be possible to monitor GPI transamidase activity in the absence of GPI, unless, as has been suggested(44) , enzymatically active transamidase exists as a GPIbulletenzyme complex.


Figure 7: Summary of the processing of prominiPLAP in RM in the presence of different nucleophiles.




FOOTNOTES

*
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 correspondence should be addressed.

(^1)
The abbreviations used are: GPI, glycosylphosphatidylinositol; site, the amino acid in a nascent protein that accepts the GPI moiety and, after cleavage, becomes the COOH-terminal residue of the mature protein; RM, rough microsomal membranes; ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-phospholipase C; PLAP, placental alkaline phosphatase (human); VSG, variant surface glycoprotein; PAGE, polyacrylamide gel electrophoresis; TEA, triethanolamine.

(^2)
Vidugiriene, J., and Menon, A. K. (1995) EMBO J.14, in press.


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