Int. J. Peptide Protein Rex 25,1985,9-14

Evidence for the existence of [Gln9 ] 4-lipotropin in human pituitary glands

DAVID CHUNG and CHOH H A 0 LI

Laboratory of Molecular Endocrinology, University of California, San Francisco, CA, USA

Received 5 April, accepted for publication 17 May 1984

The isolation of two peptides similar in amino acid composition t o that of human 0-lipotropin is presented. Peptide patterns after enzymatic digestions of these t w o peptides by Staphylococcus aureus protease and by trypsin were nearly identical. Paper electrophoresis and amino acid analyses of acidic peptides generated from the enzymatic digestions of these two peptides indicate that there is an amide difference between the two peptides. It is proposed that this amide difference is in amino acid residue number nine, and that one is the human 0-lipotropin and the other its [Gln9] analog.

Key words: HPLC; paper electrophoresis; peptide pattern; S. aureus protease; trypsin

The primary structure of human 0-lipotropin (Ph-LPH) has been the focus of several publi- cations since the initial proposal in 1976 (1-8). The accepted amino acid sequence is that deduced from the revised nucleic acid sequence of the precursor gene ( 5 ) , and by the direct amino acid sequencing of the flh-LPH (6-8). The final proof o f the accepted primary structure of oh-LPH (see Fig. 1) is achieved by the total synthesis of the hormone (9) .

During the course of investigating an improved procedure for the isolation of human pituitary peptides, two Ph-LPHs were isolated, differing from each other by one net electrical charge. The evidence presented herein indicates that the difference between the two Ph-LPHs is a Glu/Gln interchange in residue position 9 o f Ph-LPH (see Fig. 1).

Abbreviations: LPH, lipotropin; Ph-LpH, human p-LPH; RP-HPLC, reverse-phase high performance liquid chromatography; CMC, carboxymethyl cellulose.

H-GLU-LEU-THR-GLY-GLN-ARC-LEU-ARG-GLU-GLY-ASP-GLV-PRO-ASP-GLY- 5 10 1 5

PRO-Au-ASP- 4s~-GLY-ALA-GLY-Au-GLN-Am-~SP-LEU-GLU-H I S-SER- 20 25 30

LEU-LEU-VAL-ALA-ALA-GLU-LYS-LYS-ASP-GLU-GLV-PRO-TVR-ARG-~ET- 3 5 60 4 5

GLU-HI S-PHE-ARG-TRP-GLY-SER-PRO-PRO-LYS-ASP-LVS-ARG-TYR-GLV- 50 55 60

GLY-PHE-MET-THR-SER-GLU-LVS-SER-GLN-THR-PRO-LEU-VAL-THR-LEU- 6 5 70 75

PHE-LVS-ASN-AM- ILE-ILE-LVS-PSN-ALA-TVR-LVS-LYS-GLV-GLU-CH~ 80 8 5 89

FIGURE 1 The amino acid sequence of ph-LPH.

EXPERIMENTAL PROCEDURES

Four hundred fresh frozen human pituitaries, kindly provided by the National Pituitary Agency, were processed as described ( 2 ) using acid-acetone extraction, Sephadex (3-10

9

D. Chung and C.H. Li

desalting, CMC chromatography, Sephadex G-50 chromatography. The final step in the isolation was a reverse-phase HPLC which was performed in a 4.5 x 250mm Alltech Vydac 201TP column with a linear gradient of 25-40% 2-propanol in 0.1% trifluoroacetic acid. Detection was carried out at 280nm or 210nm.

Monitoring of all fractions were performed by paper electrophoresis and amino acid analyses. Paper electrophoresis was run on Whatman 3MM paper in a pH 6.7 buffer (7-collidine: acetic acid:H20, 8.9: 3.1:988, U/U) at 400V for 2 h. Amino acid analyses. were performed according to the method of Spackman et al. (10) in an automatic amino acid analyzer (Model 119C, Beckman Instruments). NH2 -terminal residue was deter- mined by the dansyl method (1 1).

The enzymes used were: trypsin (Worthington TPCK, 30A872), Staphylococcus aureus protease (Miles Laboratories, 36.900-0877) and leucine aminopeptidase (Worthington, 55B411).

RESULTS

The CMC chromatogram of 835 mg Fraction D is shown in Fig. 2. Components E and F were then further fractionated by Sephadex G-50

OM hl 0.IU 02u 0.SY N b A C

4 300

Tube number

FlGURE 2 The CMC chromatography of 855 mg Fraction D. The column dimensions are 2 X 42 cm and it is packed with Schleicher and Schuell CM cellulose. The elution is by a continuous gradient with a 500ml mixing chamber initially filled with 0.01 M NH,OAc, pH 4.6. At tube 30, 0.1 M NH,OAc, pH 7.0, is introduced to the mixing chamber, and at tube 130,0.2M NH,OAc, pH 7.0 is introduced. Detection of effluent is at 280 nm. As designated, the fractions are pooled, lyophilized and re-lyophilized twice to remove the NH,OAc.

10

FIGURE 3 The Sephadex C-50 chromatographies of Fractions CMC-E and CMC-F. Fraction CMC-E, 50mg and CMC-I:, 28 mg, are applied to a 2.5 X 75 cm column of Sephadex G-50 (fine) equilibrated with 0.01 M NH,OAc, pH 4.6. I:low rate was 30ml/h, 3ml/tube fractions were collected and detection was at 280 nm. The yields were 38mg and 15mg of G-SO-E and G-5 0-1:.

chromatography (Fig. 3). The material with Ve/Vo of approximately 1.3 was finally purified by preparative RP-HPLC (Fig. 4). Fig. 5 is the analytic RP-HPLC of purified E and F. The overall yields were: E, 25mg; F, 9.5 mg.

F

- - 0 10 M 30 0 10 20 30

Tlnu(min)

FIGURE 4 The preparative RP-HPLC of G-50-E and G-50-F. The column used is an Alltech Vydac 201TP, 4.5 X 250 mm. Elution is by a linear gradient of propanol-2 with 0.1% trifluoroacetic acid, 25 - 40% propanol-2 over 30mm, 0.5 ml/min flow rate. Sample load is approximately 3mg each of G-50-E and C-50-F. Detection is at 280 nm with a Laboratory Data Control (LDC) Spectromonitor 111. The pumps, LDC Con- stantmetric I and IIG, were controlled by an LDC Gradient Master. The recorder used was a Heathkit IR-5204.

[Glns]-@-LPH in human pituitary gland

TABLE 1 Amino acid compositionaof E and F

Amino acid E F Theoreticalb

Asx 9.5 9.5 9 Thr 4.0 4.1 4 Ser 4.0 3.9 4 Glx 11.8 10.9 11 Pro 5.5 5.5 6 G ~ Y 9.5 9.1 11 Ala 1.3 6.6 8 Val 2.3 2.2 2 Met 1.7 1.8 2 I le 1.2 1 . 3 2 Leu 7.0 6.5 I Tyr 2.9 2.4 3 Phe 2.9 2.5 3 His 1.8 1.6 2 LY s 8.8 8.5 9 A% 4.6 4.7 5 TrP n.d n.d 1

0 6

I I I I I I 0 10 20 0 I0 20 30

Time (mn 1

FIGURE 5 The analytical RP-HPLC of the RP-HPLC purified E and F. The column and equipment are described in Fig. 4. The linear gradient used is 20-40% over 30min, 0.5 ml/min flow rate and detection at 2lOnm. 25 lug each of RP-HPLC E and F were used.

As shown in Fig. 6, the mobility of F is more cathodic than that of E on paper electro- phoresis at pH 6.7. The amino acid analyses of E and F are presented in Table 1 . Not only are the amino acid compositions very similar,

00 0- I I 1 I

E E* F F*

FlCURE 6 The paper electrophoresis chromatogram of E and 1: and E* and P* treated with 0.1 M NH ,OH. The buffer is r-collidine-HOAc-H,O, pH 6.7 (8.9 ml y-collidine- 3.1 ml HOAc-988 ml H 20). Electrophoresis for 2 h at 400V and detection by ninhydrin development (0.1% nlnhydrin in ethanol).

a 1 lo", 20 h in constant boiling HCI hydrolysis. n.d., not determined. bFrom the amino acid sequence, see Fig. 1.

but both E and F have Glx as the amino terminal residue.

When both E and F are treated with 0.1 M NH40H at 37" for 1 h with a concentration of SOpg/SO pl , the electrophoretic mobility of E does not change, while treated F becomes more acidic as shown in Fig. 6. This suggests that F may have a labile amide in its primary structure. Thus the use of Staphylococcus aureus protease (S. aureus), an enzyme which is known to cleave after Asp and Glu in peptides, seems appropriate.

T l r n Imn)

FIGURE 7 The RP-HPLC of the S. aureus digests of E and F. Column and equipment as described in Fig. 4. The linear gradient was 10 .+ 30% propanol-2 over 60 min and detection was at 210 nm.

1 1

D. Chung and C.H. Li TABLE 2

Amino acid composition of S. aureuspeptidesa

Amino acid E-13A E-l2B/13B F-13A F- 1 OA F-1OB

Asx 4.7 ( 5 ) 4.3 (4) 5.1 (5) Thr 1.1 (1) 1.2 (1) Glx 2.7b(3) 2.0 (2) 2.7b(3) 2.0 (2) 2.0 (2) Pro 1.3 (2) 2.0 (2) 2.0 (2) GlY 1.0 (1) 3.8b(S) l .O-(l) 3.6b(4) 4.495) Ala 3.9 (4) 3.6 (4) 4.1 (4) Leu 2.2 (2) 1.1 (1) 2.1 (2) 0.9 (1) 1.0 (1) A rg 1.8 (2) 2.0 (2)

Ph-LPH (1-9) (10-28) (1-9) (12-28) (10-28) no. residuec

*See Fig. 8; values in parentheses are theoretical.

CSee Fig. 1. Low recovery due to ninhydrin reaction.

S. aureus, 20 pg , was added to 0.5 mg each Paper electrophoresis at pH 6.7 of the fractions E and F in 0.25 ml 0.2 M NH40Ac buffer of in the 10 min region, which exhibit dissimilarity, pH 8.2. The solutions were incubated at 37" is shown in Fig. 8. After ninhydrin development for 2 h. Glacial HOAc (10~1) was added to each and elution with dilute ammonia, the amino sample and the samples lyophilized. The digests acid analyses of the peptides are shown in were then fractionated on RP-HPLC (Fig. 7). Table 2.

Similarly, trypsin digestions were performed on E and F. The digestions were at pH 7.0 for 4 h with 1Opg trypsin and 0.5mg E or F. Experiments showed that RP-HPLC of the digests gave similar pattern (see Fig. 9). Paper electrophoresis of the 29.5 min fractions from RP-HPLC gave acidic peptides in reasonable

0

I I I I 1 1 I 1

E 12 E 13 F 10 F I3

FIGURE 8 The paper electrophoresis pattern of the lOmin regions of the RP-HPLC fractions of the S. uureus treated E and F. Conditions are as described in Fig. 6 .

12

O a r A

0 10 20 30 0 10 20 30 40

FIGURE 9 The RP-HPLC of a tryptic digest of E and F. Details are as described in Fig. 4. The linear gradient was 10 - 30% propanol-2 over 30 rnin and detection was at 210nrn.

[Gln*]-P-LPH in human pituitary gland

TABLE 3 Amino acid composition of acidic peptidesaof tryptfc digests of E and F

Amino acid E-A E-B F-A F-B F-C Theoretical

Asx Ser Glxb Pro

Ala Val Leu His

G ~ Y

Lysb

(5.7) 1.4 3.6 1.6 5.1 5.7 1.1 3 .O 1 .o 0.2

4.5 (2.4) 3.7 1.9 5 .o 5.8 1 .o 2.9 0.7 0.4

(5.6) 1 .o 3.1

(1.3) 4.1 5.5 1.0 2.9 0.8 0.2

(5.6) 1.1 3.5 1.8 4.9 5.9 1 .o 3.1 0.8 0.2

5.0 1.1 3.5 2.1 4.6

(5.0) 0.9 2.9 0.6 0.3

5 1 4 2 5 6 1 3 1 1

%ke Fig . 10. LOW values due to losses during ninhydrin reaction.

homogeneity (see Fig. 10) and their amino acid compositions are summarized in Table 3.

The acidic tryptic peptides were then treated with leucine aminopeptidase, 1 pl enzyme suspension (approx. 3 pg enzyme) was added to each peptide (approx. SOpg), in 5Opl pH 8.5 Tris buffer for 6 h at 37". Amino acid analyses of the digests show the presence of Cln in the least acidic of the acidic peptides of F (F-C, see Fig. 10).

0

I I E F

FIGURE 10 The paper electrophoresis pattern of the 29.5 min fractions after RP-HPLC (see Fig. 9) of the tryptic digest of peptides E and F. Conditions are as described in Fig. 6.

DISCUSSION

Two forms (designated as E and F) of P-LPH have been isolated from human pituitary glands. From 400 glands, 25 mg E and 9.5 mg F were obtained. E, which was employed for sequence determination (2), is ph-LPH whereas F is another form of &-LPH. The fact that treat- ment of F with 0.1 M NH40H resulted in a more acidic peptide (see Fig. 6) suggests the presence of a labile amide group. This same treatment does not alter the electrophoretic mobility of E. Trypsin and S. uureus digests of E and F yielded peptides which give similar patterns on RP-HPLC (Fig. 9) and paper- electrophoresis except for the peptides from the acidic region (residues 9-37, see Fig. 1) of Ph-LPH. This is the region of difficdty and disagreement in amino acid sequencing

As anticipated, if peptide E is the Ph-LPH with Glu9, S. mreus on E yielded peptides 1-9 and 10-28. On the other hand, S. aureus on peptide F yielded an additional peptide, residues 12-28 (see Table 2). This peptide, 12-28, could have been produced if in F, there is a Gln9. The concurrent presence of peptides 1-9 and 10-28 can indicate a rapid deami- dation of Gin9 during the enzymatic process.

Trypsin digestion of peptides E and F gave rise to multiple acidic peptides, peptides with residues 9-31 and 9-37. All of these peptides had similar amino acid compositions. The

(2,4,6-8).

13

D. Chung and C.H. Ii

analyses suggest the partial conversion of Gin9 and Gln24 during the enzymatic process. LRucine aminopeptidase digest of the acidic tryptic peptides from E and F (see Fig. 10) do reveal the presence of Gln from the least acidic tryptic peptide of F. However, Glu was also found in this case, an indication of the deamidation of the Gln9.

If one of the two &,-LPHs is artifactually produced during isolation this would necessarily mean that F is converted to E. This conversion would be most probable as there appears to be one (or more) labile amide (s) in F. Since there are no known enzyme (s) that can convert a Glu in peptide to Gln, it appears that post translational processing of E to F is not likely. Finally, the possibility of polymorphism in the precursor gene can not lie excluded as an explanation for the existence of these two oh- LPHs. Multiple genes for the precursor pro- opiomelanocortin have been reported for the rat (12, 13), porcine (14) and salmon (15). If a Glu/Gln can be explained by polymorphism, then other changes might be present, especially in the acidic region of Ph-LPH. It may be noted that in sheep pituitary glands, P-LPH exists in two polymorphic forms (16) differing by one amino acid at residue position 1 (Glu/< Glu).

In conclusion, the data presented shows the presence of two Ijh-LPHs. The difference between the two Ph-LPHs is proposed to be in amino acid residue 9, a Glu/Gln interchange. A reasonable explanation would be two different gene for the precursor pro- opiomelanocortin, which has already been detected for other species. If indeed two genes for proopiomelanocortin are present, dif- ferences other than the Glu/Gln change with residue 9 may well be present. This could possibly explain amino acid sequence dif- ferences, in the region of residues 9-37 in earlier publications (2-8).

ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Health Grant GM02907 and the Hormone Research Foundation.

REFERENCES

1 . Li, C.H. & Chung, D. (1976) Nature 260, 622- 6 24

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Biophys. Acta 451, 124-132

Address: Dr. C.H. Li Laboratory of Molecular Endocrinology 1018 HSE University o f California, San Francisco, CA 94143 USA

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