Invented by Se-Jin Lee, Alexandra C. McPherron, School of Medicine of Johns Hopkins University
The School of Medicine of Johns Hopkins University invention works as followsThe present invention consists of isolated polynucleotides which encode promyostatin or a portion thereof in polypeptide form, complementary polynucleotides, and oligonucleotides capable of hybridizing with such polynucleotides. The present invention provides a polynucleotide that encodes a mature myostatin-peptide.
Background for Polynucleotides encoding promyostatin polypeptides
The invention is a general description of peptide portions from promyostatin polypeptides (growth differentiation factor-8, GDF-8), and more specifically compositions that modulate the myostatin transduction signal in a cellular environment, as well as methods for using these compositions.
2. Background Information
The amount of money, time and effort spent by Americans each year to lose weight is staggering. Many of these people are not only trying to lose weight, but also to avoid the medical complications that come with being overweight.
More than half the adult population of the United States are overweight. Twenty to thirty percent (and thirty to forty) of American adult women and men are obese. The highest rates of obesity occur among minorities and the poor. Over the last few decades, obesity, defined as being at least twenty percent over the average level of adiposity has increased dramatically. It is now a major issue among children. Twenty percent of children are overweight today, which is a double in the last five years.
Obesity, and the medical conditions directly related to it, are major causes of morbidity in the world. Obesity increases the risk of developing various diseases, such as atherosclerosis and hypertension. It also increases the likelihood of a heart attack and type II diabetes. Type II diabetes is one of the most common causes of death in the United States.
More than 80 percent of Type II diabetes is caused by obesity. Type II diabetes is prevalent in all races but particularly among Native Americans. Type II diabetes is now more common in children than in adults. The number of reported cases has almost tripled in the last five year. Type II diabetes is also known as non-insulin dependent diabetics. It is characterized both by a reduced response of insulin to glucose, and by a resistance to insulin action, even when insulin levels are generally normal or high. Type II diabetes can affect the function of many different tissues and organs. It can also lead to vascular diseases, renal failure and neuropathy.
Unlike the medical issues associated with obesity and certain chronic diseases, severe weight loss is a common occurrence in patients who have these conditions. This presents a medical challenge. This weight loss is referred to by the term cachexia. However, its molecular cause is still not fully understood. However, it is evident that cachexia can complicate the management of these diseases and be associated with a bad prognosis. “The effects of cachexia can be seen in the wasting symptoms that occur in cancer patients and AIDS sufferers.
Despite the great effort made to understand the biological processes that regulate body weight, results have generated more hype than value. Leptin, for example, was hailed by scientists as a breakthrough for understanding the molecular foundation of fat accumulation in humans and the promise of an obesity cure. Leptin was found to be involved in the transmission of internal signals that regulate appetite in animal studies. This suggested that leptin might help treat obesity in humans. Leptin’s use in treating obesity is still a slow process. It has also not met expectations.
The treatment of morbidly overweight people is currently limited to surgery, which removes portions of intestine to reduce the amount of food and calories absorbed. The only “treatment” for moderately obese people is to eat a healthy diet and exercise regularly. Eating a healthy diet, and exercising regularly is the only?treatment? for moderately obese people. This method has shown modest success at best. There is a need to identify biological factors that regulate body weight including muscle development, fat accumulation and other disorders. This invention meets this need, and also provides additional benefits.
The present invention is a substantially pure peptide of a polypeptide promyostatin. The present invention is illustrated by a promyostatin peptide that has an amino-acid sequence similar to that of human (SEQ NO: 2), murine (SEQ NO NO: 4), rat (SEQ NO NO 6), baboon (SEQ NO 10), bovine (SEQ NO 12), porcine (SEQ NO 14), and ovine (SEQ NO 16); as well as by a polypeptide of a piscine type such as the zebrafish poly A promyostatin is also exemplified here by a polypeptide containing the portion of Salmon allele 1 (SEQID NO: 27) and the portion or Salmon allele 2(SEQID NO: 29).
The present invention also relates a to a pro-GDF polypeptide or a functional portion thereof. These proteolytic fractions are illustrated here by the proteolytic portions of a progdf-11 polypeptide or a promyostatin-like polypeptide. Pro-GDF fragments or their functional peptide portions are characterized in part by an activity related to GDF signaling. A promyostatin peptide or functional portion thereof can, therefore, have, for instance, myostatin-binding activity, myostatin-signal transduction stimulatory activity or inhibitory activity. The proteolytic fragment can be a fragment that is produced when a promyostatin peptide is cleaved at a recognition site for proteolytic cleavage having the amino acids Arg-Xaa Xaa Arg (SEQ NO: 21). These proteolytic recognition site are exemplified in the Arg Ser Arg Arg (SEQ NO: 22) amino acid sequence, which is shown in SEQ NO: 1 (promyostatin), or in the amino acid sequences 295 to 298, of SEQ NO: 25 (“human pro-GDF-11”) and the Arg Ile Arg Arg (SEQ NO: 23) amino acid sequence.
The proteolytic fragment can be a GDF prodomain. For example, it could be a myostatin prodomain which contains amino acid residues from 1 to 262 of a polypeptide promyostatin, or its functional peptide part, or even a GDF-11 prodomain which comprises amino acid residues from 1 to 295 of the pro-GDF-11 peptide or its functional peptide part. The amino acid residues of a myostatin are shown in SEQ NO. 4 and SEQ NO. 6, as well as in SEQ NO. 2, SEQ NO. 10, SEQ NO. 12, SEQ NO. 8, SEQ NO. 18, SEQ NO. 14, SEQ NO. 16, SEQ NO. 20. A functional peptide of a prodomain of myostatin is illustrated by a portion of the prodomain of myostatin that interacts specifically with myostatin, or promyostatin. A GDF-11 prodomain can be exemplified as amino acid residues 1 – 295 in SEQ ID No: 25. A functional peptide of a GDF-11 prodomain can be exemplified as a peptide of a GDF-11 prodomain which interacts specifically with mature GDF-11, or a pro GDF-11 polypeptide. The functional peptide of a GDF-prodomain should reduce or inhibit the ability of that GDF or related GDFs to stimulate signal transduction.
In a further embodiment, a proteolytic fragment is a mature GDF polypeptide or a functional portion thereof. The proteolytic fragment may be a mature C terminal GDF-11 or myostatin, which contains about amino acids residues from 268 to 374. The amino acid sequences 268-374 of a mature myostatin are shown in SEQ NO 4 and 6; 267-374 in SEQ NO 2, SEQ NO 10, SEQ NO 12, SEQ NO 8, SEQ NO 16, SEQ NO 20, and the amino acid sequences 27 and 29. A functional peptide of a mature myostatin can be exemplified as a peptide of myostatin with myostatin-signal transduction stimulating or inhibiting activity. A mature GDF-11 is represented by the amino acid residues of SEQ ID No: 25. A functional peptide of a mature GDF-11 is represented by a portion of mature GDF-11 with GDF-11 signal-transduction stimulating or inhibiting activity. The functional peptide of a GDF-11 peptide may have, for instance, the ability to interact with a receptor specifically, the ability to reduce or block the ability of the mature GDF-11 peptide interact with its specific receptor, or other activities that stimulate or inhibit GDF signaling transduction activity.
The present invention also relates to mutant pro GDF polypeptides, such as mutant promyostatin or mutant pro GDF-11 polypeptides, that contain an amino acid substitution that disrupts proteolyticcleavage in a proteolyticcleavage site with the amino acid sequence of Arg-Xaa, Xaa, Arg (SEQ NO: 21). A mutant promyostatin polypeptide or pro-GDF-11 can contain a mutation in an Arg residue from SEQ ID No: 21 such that it cannot be cleaved to a prodomain or a mature GDF peptide. The mutant GDF peptide should have a dominant-negative activity in comparison to the wild type GDF peptide. “For example, a pro-GDF-11 mutant polypeptide, or a promyostatin mutant polypeptide, can show a dominant negative effect on myostatin, or GDF-11.
The present invention also pertains to a polynucleotide that encodes a peptide part of a pro-GDF-11 or promyostatin polypeptide. The polynucleotide could encode, for example, a GDF-11 or myostatin prodomain or a functional portion thereof. The invention also relates to antibodies which can specifically bind a peptide part of a polypeptide promyostatin, such as a myostatin prodomain, or a functional portion thereof, and to antibodies which can specifically bind a pro-GDF-11 peptide. Kits containing a mutant promyostatin, a pro GDF-11 or a pro-GDF-11, a polynucleotide that encodes a pro GDF-11 or a pro peptide are also provided.
Also, the present invention is a method for identifying a functional portion of a GDF-11 or myostatin-prodomain that interacts with a GDF-11 or myostatin-peptide or both. The invention can be carried out, for instance, by testing the ability of a peptide of a GDF-11 or myostatin-prodomain to interact with myostatin, and then detecting the specific interaction between the peptide part and the myostatin. In one embodiment, the method of the invention can be performed on a computer by testing the ability of a virtual portion of a GDF-11 or myostatin protodomain to interact specifically with a virtual virtual myostatin. In a second embodiment, the method involves contacting the peptide of the prodomain with the myostatin under conditions that allow a prodomain specifically to interact with a peptide.
The present invention provides a substantially pure peptide of a polypeptide promyostatin. The prodomain of the myostatin mature peptide, previously known as GDF-8 (growth differentiation factor-8), is located at its amino-terminal. No. 5,827,733). The mature myostatin is responsible for the activity of myostatin after its cleavage. Promyostatin, then, is a polypeptide precursor that is proteolytically broken down to produce myostatin. The myostatin protodomain, as disclosed herein can inhibit GDF-11 activity or myostatin.
The present invention also provides an essentially purified peptide component of a pro GDF-11 polypeptide. Pro-GDF-11 (previously referred to as GDF-11) comprises an amino-terminal prodomain and a mature GDF-11 C-terminal peptide. The WO 98/35019 is incorporated by reference in this document. The mature GDF-11 is responsible for the GDF-11’s activity after its cleavage. Pro-GDF-11 is, therefore, a precursor polypeptide, similar to promyostatin. It is proteolytically broken down to yield active GDF-11. The GDF-11 prodomain, as disclosed herein can inhibit GDF-11 or myostatin activities, or both.
The transforming growth factor (TGF) is composed of promyostatin, pro-GDF-11 and other members. (TGF-?) The TGF-? superfamily is a group of polypeptides with multifunctional functions that regulate proliferation, differentiation and other functions within various cell types. The TGF? The TGF-? Biophys. Res. Comm. Chem. Chem. 265:13198, 1990). The TGF? The TGF-? Rev. Biochem. Biochem.
Many members of the TGF family have regulatory effects (positive or negative) on other peptide growth factors. “Many of the TGF-? Specifically, some members of the TGF? TGF-? Inhibins, activins, and other superfamily members are expressed in the nervous system (Meunier, et.al., Proceedings of the National Academy of Sciences, 1996). Natl. Acad. Sci., USA 84:247, 1989; Sawchenko and al. Nature 334:615, 1987), activin is a molecule that can be used to survive nerve cells (Schubert et. al. Nature 344:868, 1988). Growth differentiation factor-1, another member of the family, is expressed in a way that is specific to the nervous system (Lee, Proceedings of the National Academy, 1988). Natl. Acad. Sci., USA 88 : 4250, 1991), as well as other members of the family such Vgr-1, (Lyons et al. Natl. Acad. Sci. USA 86:4554, 1989; Jones et al., Development 111:531, 1991), OP-1 (Ozkaynak et al., J. Biol. Chem. The nervous system also expresses BMP-4, 267:25220 (Jones, Development 111, 531, 1991), and BMP-2 (Brown, Trends Neurosci. Brown, Trends Neurosci.) has shown that skeletal muscles produce a factor (or factors) that promotes the survival of motor neurones. The expression of GDF-8 and GDF-11 (myostatin) in muscle suggests myostatin or GDF-11 may be neurotrophic factors. Methods for modulating myostatin or GDF-11 activity, or both, can be used to treat neurodegenerative disorders such as muscular dystrophy or amyotrophic-lateral sclerosis, or maintain cells or tissues in cultures prior to transplantation.Click here to view the patent on Google Patents.