Amino-terminal amino acid sequence and carboxyl-terminal analysis of Rauscher murine leukemia virus glycoproteins

VIROLOGY

85, 319-322

(1978)

Amino-Terminal Amino Acid Sequence and Carboxyl-Terminal Analysis of Rauscher Murine Leukemia Virus Glycoproteins LOUIS

Viral

E. HENDERSON, TERRY HANS MARQUARDT, Oncology

Program,

NCI

Frederick Accepted

D. COPELAND, AND STEPHEN Cancer

Research

November

Center,

GARY W. SMYTHERS, OROSZLAN’ Frederick,

Maryland

21701

7,1977

The two glycoproteins of Rauscher murine leukemia virus, gp70 and gp45, were found to have the following identical NH,-terminal amino acid sequences: Ala-AlaPro-Gly-Ser-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-Val(X - unidentified). The COOH-terminal amino acid of gp70 is tyrosine and that of gp45 is leucine. Computeranalyzed amino acid compositional data indicated that the protein moiety of gp45 is smaller than that of gp70. These results are compatible with the suggestion that gp45 is derived from gp70 by proteolytic cleavage.

Available information suggests that the enu gene of Rauscher murine leukemia virus (R-MuLV) codes for the polypeptide moiety of an 85,000-90,000 molecular weight glycosylated precursor protein (gPr85”“‘) which, by subsequent proteolytic cleavage, gives rise to gp70 and p15(E) viral envelope antigens (l-4) (Schultz and Oroszlan, unpublished). In purified murine type C viruses, in addition to gp70, a smaller glycosylated protein, gp45, has also been detected in variable amounts (5-8). The origin and nature of gp45 remained unknown until quite recently. One assumption was that it may be derived from cellular host components. Actin, for example, has been observed in purified virus (6). It has also been suggested that gp45 may be related to mouse H-2 antigen (9). The latter was recently shown to be an integral part of the virion envelope by immunological methods (9, 10). In recent studies, chemical and immunological evidence was presented to show that gp70 and gp45 of R-MuLV are highly related glycoproteins (11, 12). A similar conclusion has been made based on tryptic fingerprint analysis (13). In addition, immune precipitation with antiserum to pu1 Author addressed.

to whom

requests

for reprints

should

rified gp70 or natural mouse sera and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) always showed the presence of both glycoproteins in purified virus; however, similar techniques usually revealed the presence of only gp70 and gPr85 in virus-producing cells (1-4, 12, 14) (Schultz and Oroszlan, unpublished). These findings suggested a viral-specific nature of gp45, on one hand, and the possibility of involvement of proteolytic cleavage in producing gp45 from gp70 after virus maturation and budding, on the other. A recent report indicated that such enzymatic cleavage can take place in vitro (15). In this communication, we present the results of amino acid analyses, NH,-terminal sequence determinations, and carboxypeptidase digestions which clearly show that: (a) gp70 and gp45 have common NH,-terminal amino acid sequences; (b) the protein component in gp45 is smaller than that in gp70; and (c) the two glycoproteins have different COOH-terminal amino acids. R-MuLV gp 70 and gp45 were purified as described previously (11). Protein samples for amino acid analysis were hydrolyzed in uucuo at 110” for 24, 48, and 72 hr with 6 N HCl containing 0.1% phenol.

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0042-6822/78/0851-031%$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Amino acid analyses were performed on a Beckman 121 M automatic amino acid analyzer operated in the single-column mode and monitored with an Autolab-Systern AA computing integrator (16). The analyzer was modified to maintain the ninhydrin reaction coil temperature at 150”. The ninhydrin reagent contained 1% ninhydrin and 0.15% hydrandantin in pyridine:acetic acid:water (12:5:3 v:v). This reagent, which has a good buffer capacity, is stable for weeks under N, at 4” and prevents precipitate formation, a frequently encountered problem in conventional systems. With the above modifications, maximal color development with ninhydrin was obtained for all the amino acids (Henderson, unpublished). The amino acid analysis data were analyzed by the NIH DEC-10 computer using a program similar to that described by Boyer et al. (17). In general, the computer selects a minimum molecular weight for a polypeptide chain based only on the amino acid analysis data. The selection is made in such a way as to minimize the departure from integral values for all residues. The amino acid compositions of R-MuLV gp70 and gp45 are given in Table 1. These results are in good agreement with previous analyses except that the molecular weight directly computed from the amino acid analyzer data for the protein component of gp45 (approximately 36,000) is significantly smaller than the previously reported value (43,500) based on apparent molecular weight and mass determination (11). The calculated amino acid residue number of gp45 can be subtracted in all cases from the corresponding values for gp70 to yield zero or positive integers. These data suggested that gp45 may be derived from gp70 via proteolytic cleavage involving the loss of about 90 amino acid residues. In order to characterize further the two glycoproteins structurally, NH,-terminal sequence analysis and carboxypeptidase digestion were carried out as described below. NH,-Terminal amino acid sequence determinations were performed by automated Edman degradation (18) in a Beck-

TABLE AMINO

Amino acid

ACID

LYS Ax Total”

OF

gp45” Residues per protein

Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu Tv Phe His

1

ANALYSIS

33.73 28.44 32.66 23.81 47.23 31.99 17.93 17.01 2.12 9.17 29.11 11.72 5.81 8.13 10.53 14.21

gp70

gp45

AND

gp70* Nearest integer 34 28 33 24 47 32 18 17 2 9 29 12 6 8 11 14 324

Residues per protein 38.50 44.77 37.05 30.09 53.68 37.72 24.57 28.33 2.12 8.70 40.71 20.02 6.64 11.00 15.16 17.32

Nearest integer 39 45 37 30 54 38 25 28 2 9 41 20 7 11 15 17 418

a Normalized results from four independent analyses of samples hydrolyzed for 24, 24, 48, and 72 hr. * Normalized results from duplicate samples hydrolyzed for 24, 48, and 72 hr. c Without cysteine and tryptophan, values for these amino acids can be found in a previous report (11).

man Model 890 B sequenator. A microsequencing technique (19) requiring 4-8 nmol of protein was modified as previously described (20). Radiolabeled l14Clphenylisothiocyanate (PITC, specific activity: 0.2-1.2 &i/pmol) was used with dimethylallylamine (DMAA)-trifluoroacetic acid (TFA) buffer, pH 9.5, in a single coupling-single cleavage program (Beckman Instruments, Inc.). The thiazolinone derivatives of amino acids were converted to the phenylthiohydantoin (PTH) derivatives, which were extracted into ethyl acetate and identified whenever possible by three independent methods: (a) gas-liquid chromatography, glc (21); (b) amino acid analysis affer HI-catalyzed hydrolysis (22); and (c) two-dimensional thin-layer chromatography, tic (23) combined with autoradiography. The standard PTH-amino acids cochromatographed with the sample were visualized under a uv lamp and the spots were circled. The plates were placed

SHORT

Digestion with carboxypeptidase was carried out at room temperature using 0.15-0.35 nmol of protein, 0.4-0.8 pg of DFP-carboxypeptidase A, and/or 0.7-1.5 pg of PMSF-carboxypeptidase B (both enzymes obtained from Worthington Biochemicals, Freehold, N.J.) in 0.1 ml of 0.05 M NaHCO,, pH 8.3, containing 0.05% SDS. The amino acids liberated were determined on the amino acid analyzer. Serine, glutamine, and asparagine were not resolved. Digestion of gp70 with carboxypeptidase A alone for 4 hr liberated tyrosine (approximately 1 mol/mol of protein) only. A similar digestion with the mixture of carboxypeptidases A and B yielded tyrosine, arginine, histidine, and lysine (each in an approximate yield of 1 mob

in contact with Kodak X-Omat RP film for autoradiographic development. Twenty to sixty hours of exposure were generally required to locate adequately the 14C-labeled PTH derivatives. The water phase was analyzed for PTH-histidine and PTHarginine by method b only. Sequence analysis of reduced (with 2mercaptoethanol) and carboxymethylated gp70 was carried out with and without prior treatment with glycosidases to partially remove carbohydrate. The enzyme mixture prepared from Diplococcus pneumoniae was received from Dr. G. Ashwell (NIAMDD, NIH, Bethesda, Md.) It contained neuraminidase (2.0 units/ml), /3-galactosidase (4.6 units/ml), /3-N-acetylglucosaminidase (10 units/ml), and an undetermined amount of endoglycosidases and was shown to be free of proteinases (24) (Ashwell, personal communication). Our NH,-terminal amino acid analysis by the dansyl method (25) and the stepwise Edman degradation of enzyme-treated and untreated gp70 as reported here showed essentially identical results. The removal of the bulk of carbohydrate, however, resulted in cleaner glc and tic patterns of the sequencer fractions, and penetration further into the molecule was possible with enzyme-digested gp70. Analysis of gp45 was performed without previous treatment with glycosidases. Figure 1 shows a semilogarithmic plot of nanomoles of PTH derivatives of amino acids recovered at each cycle of the sequenator run with gp45. Similar repetitive yields were obtained for gp70. In Table 2 the alignment of the NH,-terminal amino acid sequences of the two glycoproteins is shown. They have identical sequences up to residue 11 and probably beyond. Residues 12 and 15 could not be identified in the present studies for either of the glycoproteins.

6.0 6.0 5.0 5.0

AMINO

ACID

SEQUENCE

AND

NH,-Terminal 1 gp79 gp45 a X, Unidentified.

5

Input

4.0

G G 3.0

,q P A

2.0 ; -

\

P

l.O0.9 0.6 0.7 0.6 0.5 o.4

E-V I 1

I

I 3

I

ss,,,,,,,,,,, 5 7 Residue

9 11 Number

13

15

17’

FIG. 1. Yields of amino acids obtained by automated Edman degradation of 4.2 nmol of gp45 and HI hydrolysis of the PTH derivates [WIPTH-alanine and [WIPTH-serine were quantitatively determined by tic plus scintillation counting (19). PTH-Alanine was quantitated by glc, also. The single letter code used for the amino acids is: A, alanine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; P, proline; Q, glutamine; S, serine; T, threonine; V, valine; Y, tyrosine.

TABLE ~~~~~~~~~~~~

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2

COOH

TERMINAL

OF

R-MuLV

sequence 10

Ala-Ala-Pro-Gly-Ser-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-ValAla-Ala-Pro-Gly-Ser-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-Val-

gp70

AND gp45’ COOH

terminal

15 -Tyr -Leu

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mol of protein). In contrast, carboxypeptidase A initially released leucine from gp45. Longer digestion with the enzyme mixture liberated leucine and either serine, glutamine, or asparagine. Thus, the COOH-terminal amino acid is tyrosine for gp70 and leucine for gp45 and the sequences also show apparent differences. The chemical data presented here clearly show that the smaller glycoprotein, gp45, present in purified R-MuLV is not actin or H-2 antigen of the mouse, neither is it another host cell-derived glycoprotein. The NH,-terminal amino acid sequence identity of gp70 and gp45 provides definitive chemical evidence for the presence of structurally highly related protein moieties in the two viral glycoproteins. This and the COOH-terminal amino acid differences taken together with the amino acid compositional data provide compelling evidence that gp45, which also contains less carbohydrate (111, is derived from gp70 through proteolysis which may or may not be accompanied by glycolytic cleavage(s). The alternative that gp45 may be generated from the nonglycosylated or partially glycosylated precursor cannot be completely excluded. In summary, the results suggest that the two glycoproteins are not separately encoded in the viral genome. ACKNOWLEDGMENT This work was Program, Contract Cancer Institute, Bethesda, Maryland

7.

8. 9. 10.

11. 12.

13.

14.

15. 16.

17. 18. 19.

supported by the Virus Cancer No. NOl-CO-25423, National National Institutes of Health, 20014. REFERENCES

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