WO1989000167A1

WO1989000167A1 - Proteins and protein compositions and their use

Info

Publication number: WO1989000167A1
Application number: PCT/SE1988/000361
Authority: WO
Inventors: Tore Curstedt; Bengt Robertsson; Hans Jörnvall
Original assignee: Kabigen Ab
Priority date: 1987-07-01
Filing date: 1988-06-29
Publication date: 1989-01-12
Also published as: AU1996388A

Abstract

A protein comprising the amino acid sequence (I) wherein K and L are selected from Ile, Gly, Val, and wherein preferably K is Ile and L is Gly or both are Val; protein compositions and pharmaceutical compositions; such protein or composition for use as components of a pulmonary surfactant; and a method of facilitating respiration in mammals including man.

Description

Proteins and protein compositions and their use.

The present invention relates to proteins and protein compositions useful as components of pulmonary surfactants, i.e. for providing for normal respiration in mammals including man.

Pulmonary surfactant, which is a phospholipid-prot ein complex, is essential for normal respiration by reducing surface tension at the air-liquid interface of the alveoli (1). Different surfactant-specific proteins have been detected. One group comprises comparatively large glycoproteins with molecular weights varying between 28 and 36 kDa, depending on the degree of glycosylation (2,3). This protein is soluble in water and the primary structure of the forms from canine and human lung has been determined (4-6). In the presence of calcium ions this protein apparently participates in the formation of surface-active tubular myelin from secreted lamellar bodies (7) and increases the rate of adsorption of surfactant phospholipids (8). Although this protein probably is functional for the endogenous surfactant, synthesized in the alveolar epithelial type II cells, it does not seem to be essential for the physiological activity of exogenous surfactant preparations designed for replacement therapy (9-11).

A second group of surfactant-specific proteins constitutes forms with low-molecular weights (≤14 kDa) (2, 9, 12-20). These proteins are very hydrophobic and are composed of different proteins which may be soluble (9) or insoluble (2,16,17) in ether/ethanol. Both proteins require organic solvents for solubilization and purification, and are heterogenous by multiple start positions in the N-terminal regions (truncated forms). Recombination of either of these proteins with synthetic phospholipids yields a surfactant preparation with physical and biological properties which in many respects are similar to those of natural pulmonary surfactant. The two low-molecular weight proteins have unrelated structures and sizes; the smaller form is not a fragment of the larger. Recently, cDNA segments of the longer form (21) have been described from dog, but exact structures, sizes or further properties have not been defined. The present invention concerns lypophilic low-molecular weight apoproteins of mammalian origin.

Based on extensive scientific research and experimentation a new protein has been found possessing pulmonary surfactant activity, said protein comprising the amino acid sequence:

15 20 25

Leu-Leu-Val-Val-Val-Val-Val-Val-Leu-Leu-Val-Val-Val-K-Ile- 30 35 L-Gly-Ala-Leu-Leu-Met-Gly-Leu, (I) werein K and L are selected from lie, Gly, Val, and wherein preferably K is lle and L is Gly or both are Val.

A preferred protein comprising said amino acid sequence has the following structure: 1 5 10 15

M-Arg-lle-Pro-Cys-Cys-Pro-Val-N-Leu-Lys-Arg-Leu-Leu-Val-Val-

20 25 30

Val-Val-Val-Val-Leu-Leu-Val-Val-Val-K-Ile-L-Gly-Ala-Leu- 35 Leu-Met-Gly-Leu (II) wherein M is selected from Leu and Phe and N is selected from Asn and His, and wherein K and L have the meaning given above.

In the structure of sequence II it is preferred that either M is Phe and N is His or M is Leu and N is Asn. According to another aspect of the invention there is provided a protein comprising the amino acid sequence: 10 Phe-Pro-Ile-Pro-Leu-Pro-Phe-Cys-Trp-Leu-Cys-Arg-Thr-Leu-Ile-

20 30

Lys-Arg-Ile-Gln-Ala-Val-Val-Pro-Lys-Gly-Val-Leu-Leu-Lys-Ala- 40

Val-Ala-Gln-Val-Cys-Ser-Val-Ser-Pro-Leu-Val-Val-GLy-Gly-Ile-

50 60

Cys-Gln-Cys-Leu-Ala-Glu-Arg-Tyr-Ile-Val-Ile-Cys-Leu-Asn-Met-

70 Leu-Leu-Asp-Arg-Thr-Leu-Pro-Gin-Leu-Val-Cys-Gly-Leu-Val-Leu-

Arg-Cys-Ser-Ser. (Ill)

According to yet another aspect of the invention there are provided protein compositions comprising protein I in combination with protein III and, alternatively, protein II in combination with protein III.

The present invention also involves pharmaceutical compositions comprising in combination a protein or protein composition as defined above and a phospholipid type of material. In such pharmaceutical composition the protein is a minor component, and a preferred weight range of the contents of the composition of the protein is about 0.5 to about 10% by weight thereof. It is particularly preferred that the protein constitutes about 1 to 5% by weight of the composition as a whole. As an example of phospholipid material there may be mentioned phospholipids based on palmitic acid. In addition to such phospholipid matrix the composition of the invention may also contain other additives, such as pharmaceutically acceptable carriers or diluents, stabilising agents, and other conventionally used pharmaceutically acceptable additives.

The proteins according to the present invention have been found to contribute significantly to pulmonary surfactant activity. Accordingly, the proteins and compositions of the invention are particularly useful as components of pulmc nary surfactants. Furthermore, the invention includes a method for facilitating respiration in mammals including man, such method comprising administering an effective amount of a protein or composition according to the invention to the respiratory tract of a patient in need of treatment for respiratory disorders. By such administration it is possible to significantly reduce surface tension at the air-liquid interface of the patient's alveoli. The administration can take place directly into trachea or bronchi, but can also take place through the oral cavity by using an aerosol spray of a conventional type.

In the following the present invention will be further illustrated by non-limiting examples. This exemplification will be made with reference to the appended drawings, wherein Fig. 1 shows the complete amino acid sequence of porcine surfactant fraction 1;

Fig. 2 shows the complete amino acid sequence of porcine surfactant fraction 2;

Fig. 3 shows the complete amino acid sequence of human surfactant fraction 2;

Fig. 4 shows diagrams on surface activity of artificial surfactant preparations using the pulsating bubble technique;

Fig. 5 shows the tidal volumes as a function of time intervals and insufflation pressures at varying therapeutic regimens including treatment according to the invention and in controls; and

Fig. 6 shows representative tidal volumes in untreated and treated animals.

NOMENCLATURE

The two types of surfactant low-molecular weight protein, strictly hydrophobic and soluble in methanol/chloroform, are isolated and separated by Sephadex LH-60 chromatography: both are biologically active, structurally unrelated, and show no evidence of product relationships. The larger protein containing 79 residues constitutes fraction 1 from the Sephadex LH-60 chromatography step, and is now named low- -molecular weight surfactant protein type 1. The smaller protein containing 35 residues constitutes fraction 2, and is named low-molecular weight surfactant protein type 2. Type 1 surfactant protein has been purified from pig lung and type 2 surfactant protein has been isolated from pig lung and human bronchoalveolar lavage or amniotic fluid.

EXAMPLE 1 Isolation of porcine pulmonary surfactant.

Minced pig lungs were washed with saline and the mixture was filtered. Cells and debris were removed by centrifugation at 1000xg for 15 min at 20°C and the supernatant was centrifuged at 3000xg for 2 hrs at 4°C. The pellets were then extracted with chlorof orm/methanol 2:1 (v/v) and the lipid extract obtained was separated by reverse phase chromatography on a column of Lipidex-5000 (Packard Instruments Co., Downers Grove, IL) in a system of ethylene chloride/methanol 1:4 (v/v) (22). The phospholipid fraction was used for isolation of proteins.

Isolation of hydrophobic low-molecular weight proteins. The phospholipid fraction (300-500 mg) was dissolved in 5 ml chlorof orm/methanol 1:1 (v/v) and applied to a column (40x5 cm) of Sephadex LH-60 in the system chloroform/methanol 1:1 (v/v), containing 5% 0.1 M HCl (23). The fraction, 0-470 ml, corresponding to 60% of the column volume, was evaporated to dryness and used for isolation of the proteins.

Separation of two hydrophobic low-molecular weight proteins. The material in the pooled protein fractions from several preparations (about 100-150 mg) was applied to a column (80x2.5 cm) of Sephadex LH-60 in the system mentioned above. Fractions of 5 ml were collected and weighed after evaporation to dryness. The material in the two first peaks as detected by weight determinations was analyzed and referred to as protein fractions 1 and 2, respectively.

The proteins were also separated by HPLC on a 5μm column (25 cm x 4.4 mm) of Nucleosil C _{1 8} in methanol, containing 0.1% trif luoroacetic acid. The proteins, applied in 10μlchloroform/methanol 1:2 (v/v) were detected at 214 nm. The two peaks obtained corresponded to those from the Sephadex LH-60 column.

EHXAMPLE 2 Isolation of human low molecular weight surfactant protein. Bronchoalveolar lavage

Bronchoalveolar lavage (BAD on humans was carried out with a flexible bronchoscope under local anesthesia. The bronchoscope was wedged in a middle lobe bronchus and sterile saline solution at 37°C was instilled in aliquotε of 50 ml.

The total volume instilled varied between 200 and 300 ml. The fluid was gently suctioned back after each instillation and collected in a siliconized bottle kept on ice. Immediately after completion of the lavate the bottle was transported to the laboratory.

The recovered BAL fluid was strained through a double layer of Dacron nets and the volume was measured. It was centrifuged at 400 g at 4°C for 5 min and the supernatant was stored at -20°C until further analyzed. Amniotic fluid.

Human amniotic fluid, obtained from full term pregnancies at Caesarian sections and vaginal deliveries, was filtered through a net and the volume was measured and the material was stored at -20°C until further analyzed. Isolation of hydrophobic proteins from bronchoalveolar and amniotic fluids.

To 300 ml of amniotic or BAL fluids 400 ml of methanol was added and the solution was mixed by shaking and ultraso- nication. 800 ml of chloroform was added and the mixture was shaken. After filtration the lower phase was evaporated to dryness and the phospholipid fraction which also contains the hydrophobic proteins was isolated by reverse phase chromatograph on Lipidex-5000 in a system of ethylene chloride/me thanol 1:4 (v/v).

EXAMPLE 3

Analysis of surfactant proteins.

For determination of ammo acid compositions, the proteins were reduced with dithioerythritσl (about 30 nmol/nmol peptide; 37°C; 2h) and carboxymethylated by addition of neutralized iodo (¹⁴C) acetate (120 nmol/nmol peptide; 37°C; 2h) in 8 M urea, 0.4 M Tris/HCXl, 2mM EDTA. Excess reagents were removed by exclusion chromatography on Sephadex LH-60 (40x1.1 cm) in chloroform/methanol 1:1 (v/v) containing 5% 0.1 M HCl. For analysis of ammo acid compositions, samples were hyrdolyzed in evacuated tubes for 24 h at 110°C and for 72 and 120 hrs, respectively, at 150ºC, with 6 M HCl containing 0.5% phenol. Total amino acid compositions of human and pig surfactant 2 are illustrated in Table 1. Liberated amino acids were analyzed with a Beckman 120 M instrument. The apparent molecular weights were determined by

SDS/polyacrylamide gel electrophoresis (using a 10% gel containing 8 M urea) (24). Molecular weight markers were purchased from BDH Chemicals Ltd (England) and consisted of horse- -heart myoglobin, cleaved by cyanogen bromide. Phosphorus was analyzed according to Bartlett (25).

The total amount of fatty acids was determined by boiling the fractions for 10 mm in 10% boron trifluoride in methanol (25). The unesterified fatty acids were determined by reaction of the fractions with diazomethane. The amount of different fatty acids was analyzed by gas-liquid chromatography using a 25 m fused silica capillary column coated with OV-1.

Preparations for sequence analysis were applied in chloroform/methanol 1:2. Structural analysis

The protein fractions were reduced by treatment with dithiothreitol (30 nmol/nmol polypeptide) at 37°C for 2 h, under nitrogen. The reduced samples were then ¹⁴C-carboxymethylated by treatment with neutralized lodo (¹⁴C)-acetic acid (120 nmol/nmol polypeptide; 37°C; 2 h) and purified by exclusion chromatography on Sephadex LH-60.

Samples for sequence analysis of the ¹⁴C-carboxymethylated protein were removed after solubilization in chloroform/methanol. Samples for cleavages with pepsin were dissolved in 100% formic acid, diluted to 5% formic acid, and then submitted to the enzyme (1:30, enzyme to substrate ratio;

37°C; 2 h). The peptic peptides were separated by high-performance liquid chromatography on an Ultropac C-18 column m 0.1% trifluoroacetic acid with a linear gradient of acetonit- rile. Samples for treatment with CNBr were dissolved in 100% formic acid, diluted to 70%, and then treated with CNBr (0.1 g/ml) at room temperature for 24 h. CNBr fragments were separated on Sephadex' LH-60 in chloroform/methanol, 1:1 (v/v) containing 5% 0.1 M HCl (23).

Gas-phase sequencer analysis was performed by degradation in an Applied Biosystems 470 A instrument and phenylthiohydantoin detection by reverse-phase high performance liquid chromatography using a Hewlett Packard 1090 instrument (27). Samples for liquid-phase sequencer analysis in a Beck- man 890C instrument were applied to glycine-precycled Polybrene (28), and analyzed by a similar high performance liquid chromatography system. Total compositions were obtained by hydrolysis with 6 M HCl/0.5% phenol at 100°C for 24 h in vacuum. Hydrazinolysis was performed with anhydrous hydrazine in 100°C for 6 h in evacuated tubes (29). Amino acids were quantitated with a ninhydrin-based Beckman 121M amino acid analyzer, or with a phenylthiocarbamyl-based high performance liquid chromatography system (30). N-terminal truncations of the different surfactant proteins are illustrated in Table 2. EXAMPLE 4

Recombination of isolated protein fractions with synthetic phospholipids.

1.2-Dipalmitoyl-sn-glycero-3-phosphocholin e (DPPC), 1-palmιtoyl-2-oleoyl-sn-glycero-3-phosphochol me (POPC) and

1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) were purchased from Sigma Chemical Co. (St. Louis, MO), and were used without further purification. The phospholipids were dissolved in chlorof orm/methanol 2:1 (v/v) mixed in the proportions DPPC:POPC:DPPG 55:35:10 (w/w/w) and used as the surfactant preparation "phospholipids".

To aliquots of this mixture one of the two protein fractions (assuming a protein content of 35% see Results) was added, dissolved in chlorof orm/methanol 1:2 (v/v), giving a protein to phospholipid ratio of 1:50. The solvents were evaporated to dryness and the. different surfactant preparations (phospholipids, phospholipids + protein fraction 1 and phospholipids + protein fraction 2) were suspended in saline giving a phospholipid concentration of 10 or 80 mg/ml.

EXAMPLE 5

Determination of in-vitro surface properties.

The surface properties of the protein-based artificial surfactants were analyzed with a pulsating bubble instrument (Surfactometer International, Toronto, Canada) (31). The surfactant preparations were suspended at a phospholipid concentration of 10 mg/ml, and the pressure gradient across the bubble wall was recorded at 37°C during 50% cyclic surface compression at the rate of 40/min. Surface tension was assessed at maximal and minimal bubble sizes during the 5th cycle and after 1 and 5 min of pulsation.

The results of these experiments are shown in Fig. 4. The tracings therein represent pressure gradients across the bubble wall; max and min indicate maximal (radius 0.55 mm) and minimal (radius 0.40 mm) bubble size during pulsation at a rate of 40/min. A pressure gradient close to zero at minimal bubble size corresponds to nearly zero surface tension. EXAMPLE 6

Determination of in vivo surface properties in premature newborn rabbits.

Rabbit fetuses were delivered on day 27 of gestation and tracheotomized at birth. The animals were allocated at random to the following four groups: 1, treatment with phospholipids + protein fraction 1 (n=10), 2 , treatment with phospholipids + protein fraction 2 (n=11), 3, treatment with phospholipids only (n=10), and 4, non-treated controls (n=10). Artificial surfactant was instilled via the tracheal cannula at a dose-volume of 2 ml/kg (phospholipid concentration in all preparations 80 mg/ml), before the onset of artificial ventilation. The animals were anesthetized with intraperitoneal sodium pentobarbital (0.1 ml; 6 mg/ml), paralysed with intraperitoneal pancuronium bromide (0.1 ml; 0.2 mg/ml), kept in body plethysmographs at 37°C, and subjected to pressure-controlled artificial ventilation with 100% oxygen, a frequency of 40 breaths/min, and 50% inspiration time. The insufflation pressure was first set at 35 cm H₂O for 1 min, then lowered to 25 cm H₂O for 15 min, 20 cm H₂O for 5 min, 15 cm H₂O for 5 min, and then again raised to 25 cm H₂O for 5 min (11,17). Tidal volumes (V_T) were determined with a pneumothachygraph connected to each body plethysmograph.

The experimental results are illustrated in Fig. 5 showing tidal volumes (Vt;+SEM) in animals treated with phospholipids plus protein fraction 1(●, n=10), phospholipid + protein fraction 2 (Δ, n=11), phospholipids only (■, n=10) and controls (o, n=10) at various time intervals and insufflation pressures (P_I).

EXAMPLE 7

In experiments with the same animal model as in Ex. 6 tidal volumes (VT) were recorded in control animals and in animals treated with the same artificial surfactant preparations at a concentration of 80 mg phospholipids per ml. The tracings were obtained at an insufflation pressure of 25 cm H₂O after 30 mm of ventilation.

Representative tracings are illustrated in Fig.6, showing signifiicant pulmonary surfactant activities for preparations containing the proteins of the invention as compared to phospholipids only and to controls.

REFERENCES

1. Goerke, J. (1974) Biochim.Biophys.Acta 374, 241-251.

2. King, R.J., Klass, D.J., Gikas, E.G. and Clements, J.A. (1973) Am.J.Physiol. 224., 788-795.

3. van Golde, L.M.G., Batenburg, J.J. and Robertson, B. (1987) Physiol.Rev., in press.

4. Benson, B., Hawgood, S., Shilling, J., Clements, J., Damm, D., Cordell, B. and White, R.T. (1985) Proc.Natl.Acad.Sci.USA 82, 6379-6383.

5. White, R.T., Damm, D., Miller, J., Spratt, K., Shilling, J., Hawgood, S., Benson, B. and Cordell, B. (1985) Nature 317, 361-363.

6. Floros, I., Steinbrink, R., Jacobs, K., Phelps, D., Kriz, R., Recny, M., Sultzmann, L., Jones, S., Taeusch,

H.W., Frank, H.A. and Fritsch, E.F. (1986) J.Biol.Chem. 261, 9029-9033.

7. Benson, B.J., Williams, M.S., Sueishi, K., Goerke, J. and Sargeant, T. (1984) Biochim.Biophys.Acta 793, 18-27.

8. King, R.J. and MacBeth, M.C. (1979) Biochim. Biophys .Acta 557, 86-101.

9. Whitsett, J.A., Ohning, B.L., Ross, G., Meuth, J., Weaver, T., Holm, B.A., Shapiro, D.L. and Notter, R.H. (1986) Pediatr.Res. 20, 460-467.

10. Metcalfe, I.L., Enhorning, G. and Possmayer, F. (1980) J.Appl.Physiol. 49, 34-41.

11. Berggren, P., Curstedt, T., Grossman, G., Nilsson, R. and Robertson, B. (1985) Exp.Lung Res. 8, 29-51. 12. Phizackerley, P.J.R., Town, M.-H. and Newman, G.E. (1979) Biochem.J. 183, 731-736.

13. Katyal, S.L. and Singh, G. (1979) Lab. Invest. 40, 562- 567.

14. Claypool, W.D., Jr., Chander, A. and Fisher, A.B. (1981) Fed.Proc. 40, 408. 15. Sueishi, K. and Benson, B.J. (1981) Biochim.Biophys.Acta 665, 442-453.

16. Suzuki, Y., Nakai, E., Ohkawa, K. (1982) J.Lipid.Res. 23, 53-61. 17. Suzuki, Y., Curstedt, T., Grossmann, G., Kobayashi, T., Nilsson, R., Nohan, K. and Robertson, B. (1986) Eur.J.Respir.Dis. 69, 336-345. 18. Takahashi, A. and Fujiwara, T. (1980)

Biochem.Biophys.Res. Commun. 135, 527-532. 19. Whitsett, J.A., Hull, W.M., Ohning, G., Ross, G. and Weaver, T.E. (1986) Pediatr.Res.20, 744-749.

20. Yu, S.-H. and Possmayer, F. (1986) Biochem.J. 236, 85- 89.

21. Hawgood, S., Benson, B.J., Schilling, J., Damm, D., Clements, J.A. and White, R.T. (1987)

Proc.Natl.Acad.Sci.USA 84, 66-70.

22. Curstedt, T. (1974) Biochim.Biophys.Acta 369, 173-195.

23. Bizzozero, O., Besio-Moreno, M., Pasquini, J.M. , Soto, E.P. and Gomez, C.J. (1982) J . Chromatogr. 227, 33-44. 24. Swank, R.T. & Munkres, K.D. (1971) Anal.Biochem. 39,

462-477.

25. Bartlett, G.R. (1959) J.Biol.Chem. 234, 466-468.

26. Morrison, R.W. & Smith, L.M. (1964) J.Lipid Res. 5, 600-608. 27. Hallden, G., Gafvelin, G., Mutt, V. and Jornvall, H. (1986) Arch.Biochem.Biophys. 147, 20-27.

28. Jbrnvall, H. and Philipson, L. (1980) Eur. J. Biochem. 104, 237-247.

29. Fraenkel-Conrat, H. and Tsung, CM. (1967) in Methods in Enzymology, vol. XI (C.H.W.Hirs, ed.) pp. 151-155.

30. Bergman, T. and Jbrnvall, H. (1987) in Methods in Protein Sequence Analysis (K.A. Walsh, ed.) Humana Press, Clifton, in

31. Enhorning, G. (1977) J.Appl.Physiol.43, 198-203.

Claims

CLAI MS

1. A protein comprising the amino acid sequence: 15 20 25

Leu-Leu-Val-Val-Val-Val-Val-Val-Leu-Leu-Val-Val-Val-K-Ile-

30 35

L-Gly-Ala-Leu-Leu-Met-Gly-Leu, (I) werein K and L are selected from Ile, Gly, Val, and wherein preferably K is lie and L is Gly or both are Val.

2. A protein according to claim 1 essentially consisting of the following amino acid sequence:

1 5 10 15

M-Arg-Ile-Pro-Cys-Cys-Pro-Val-N-Leu-Lys-Arg-Leu-Leu-Val-Val- 20 25 30

Val-Val-Val-Val-Leu-Leu-Val-Val-Val-K-Ile-L-Gly-Ala-Leu-

35 Leu-Met-Gly-Leu (II) wherein M is selected from Leu and Phe and N is selected from Asn and His, and wherein K and L have the meaning given in claim 1.

3. A protein according to claim 1 or 2 wherein M is Phe and N is His.

4. A protein according to claim 1 or 2 wherein M is Leu and N is Asn.

5. A protein comprising the amino acid sequence:

10 Phe-Pro-Ile-Pro-Leu-Pro-Phe-Cys-Trp-Leu-Cys-Arg-Thr-Leu-Ile-

20 30 Lys-Arg-Ile-Gln-Ala-Val-Val-Pro-Lys-Gly-Val-Leu-Leu-Lys-Ala-

40 Val-Ala-Gln-Val-Cys-Ser-Val-Ser-Pro-Leu-Val-Val-GLy-Gly-Ile-

50 60

Cys-Gln-Cys-Leu-Ala-Glu-Arg-Tyr-IIe-Val-IIe-Cys-Leu-Asn-Met- 70

Leu-Leu-Asp-Arg-Thr-Leu-Pro-Gln-Leu-Val-Cys-Gly-Leu-Val-Leu-

Arg-Cys-Ser-Ser. (Ill)

6. A protein comprising the amino acid sequence: 15 20

AAR1-AAR2-AAR3-AAR4-AAR5-AAR6-AAR7-AAR8-AAR9-AAR10-AAR11- 25 30 35 -AAR12-AAR13-K-Ile-L-Gly-Ala-Leu-Leu-Met-Gly-Leu,

wherein K and L have the meaning of claim 1 and AAR1-AAR13, inclusive, are amino acid residues selected from Val and Leu.

7. A protein according to claim 6 essentially consisting of the amino acid sequence: 1 5 10

M-Arg-Ile-Pro-Cys-Cys-Pro-Val-N-Leu-Lys-Arg-

15 20

AAR1-AAR2-AAR3-AAR4-AAR5-AAR6-AAR7-AAR8-AAR9-AAR10-AAR11- 25 30 35

-AAR12-AAR13-K-Ile-L-Gly-Ala-Leu-Leu-Met-Gly-Leu,

wherein M and N have the meaning of claim 1 and AAR1-AAR13, inclusive, have the meaning of claim 12.

8. A protein comprising the amino acid sequence:

M-Arg-Ile-Pro-Cys-Cys-Pro-Val-N-Leu-Lys-Arg-, wherein M and N have the meaning of claim 2.

9. A protein comprising the amino sequence:

1 5 10 15 M-Arg-IIe-Pro-Cys-Cys-Pro-Val-N-Leu-Lys-Arg-Leu-Leu-O-ValVal-

20 25 30 35

Val-Val-Val-P-Q-R-S-T-U-V-X-Y-Ala-Leu-Leu-Met-Gly-Leu

wherein M is selected from Leu and Phe and N is selected from Asn and His; O is selected from lie and Val; P is selected from Val, Leu and Trp; Q is selected from Val, Leu, Trp and Pro; R is selected from Leu, Val, Trp and Pro; S is selected from Trp, Val, Leu and Pro; T is selected from Pro, Val, Trp and Leu; U is selected from Val, Leu, Trp and Pro; V is selected from lie, Val, Trp, Pro and Leu; X is selected from Val, Leu, Trp and Pro; Y is selected from Gly and Pro.

10. Truncated forms of the protein according to any preceding claim possessing pulmonary surfactant activity.

11. The forms of claim 10 having N-terminai truncation.

12. The forms of claim 11, wherein the truncation comprises 1 or 2 amino acid residues.

13. A protein according to any of claims 6 to 8 in combination with the protein according to anyone of claims 1 to 5.

14. A protein composition comprising the protein according to claim 1 or any of claims 9 to 12 in combination with the protein according to claim 5.

15. A protein composition comprising the protein according to claim 2 in combination with the protein according to claim 5.

16. A pharmaceutical composition comprising in combination a protein or protein composition according to any preceding claim and a phospholipid type of material.

17. A protein, protein composition or pharmaceutical composition according to any preceding claim for use as a pulmonary surfactant.

18. A method of facilitating respiration in mammals including man, comprising administering an effective amount of a protein, protein composition or pharmaceutical composition according to any one of clams 1 to 15, to the respiratory tract of a patient subject to respiratory disorder so as to reduce surface tension at the air-liquid interface of the patient's alveoli.

19. A method according to claim 18, wherein the administration is performed directly into the trachea or bronchii.