Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-09-10
2003-03-11
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S252300, C435S320100, C435S325000, C435S006120, C536S023700, C536S023200
Reexamination Certificate
active
06531305
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods for altering fertility and more specifically to sperm-specific forms of the catalytic subunit (referred to as “C”) of cAMP-dependent protein kinases.
BACKGROUND OF THE INVENTION
The cAMP-dependent protein kinase (PKA) is a major enzyme in cellular signal transduction and is thought to mediate most of the physiological responses to cAMP in eukaryotic cells. Below a cAMP threshold concentration, PKA exists as an inactive tetramer of two catalytic (C) subunits and two regulatory (R) subunits that together can be represented as CR
2
C. The two R subunits form a dimer with each protomer attaching to the substrate-binding site of a C subunit. Some isoforms of R also associate with binding proteins collectively termed A-kinase anchoring proteins; it is believed that through these interactions PKA is targeted to specific subcellular compartments.
Activation of adenylate cyclase by extracellular signals raises the intracellular concentration of cAMP, and at a certain threshold concentration cAMP binds to the R subunits of the PKA tetramer, releasing C to phosphorylate its substrates.
There are three known genes encoding mammalian C. The C&agr; gene appears to be expressed in most tissues, including the brain, while C&bgr; gene expression is detected mainly in the brain. C&ggr; is a transcribed retroposon that has been found in primates and whose expression is detected only in testis. Proteins expressed from recombinant clones of C&agr;, C&bgr;, and C&ggr; have been shown to have distinct biochemical properties.
cAMP-dependent signaling has an important role in the control of sperm movement. Mammalian sperm are nonmotile in the testis, but as they pass through the epididymis they acquire the capacity for motility. This process is known as “epididymal maturation” and is essential for the sperm to fertilize an egg. Several studies have shown that changes in sperm cAMP levels are involved in epididymal maturation. The mechanisms by which alterations in cAMP levels lead to epididymal maturation are largely unknown.
SUMMARY OF THE INVENTION
The invention is based in part on the discovery of a novel sperm-specific form of the catalytic subunit of cAMP-dependent protein kinase referred to herein as C
s
polypeptide, or just C
s
, which is an alternatively spliced transcript of the C&agr; gene. C
s
is unique in that it has an acetylated amino terminus with an N-terminal amino acid sequence of six amino acids, which differs from the N-terminal sequence of all other previously described forms of C. The remainder of the amino acid sequence of C
s
is identical to that of C&agr;1, another protein encoded by the C&agr; gene, which was formerly referred to as the C&agr; protein.
The region of identity begins with the amino acids encoded by the beginning of the second exon of C&agr;. Thus, C
s
arises as a result of testis-specific splicing of an exon encoding the novel N-terminus to the remaining C&agr; exons. The sequence identity of the amino acids beginning with the second exon holds true in a wide variety of mammals. For example, the remainder of the sequence of ovine C
s
is identical to that of ovine C&agr;1, and the remainder of the sequence of human C
s
is identical to that of human C&agr;1.
Because C
s
is expressed only in testis cells (i.e., male cells), it provides a unique target for the diagnosis and therapy of fertility disorders, as well as a basis for novel male contraceptives and fertility enhancers. Thus, for example, nucleic acids encoding C
s
polypeptides, and agents based on these sequences, can be used for diagnosing and treating conditions associated with spermatocyte function, e.g., in the promotion or inhibition of fertility.
In general, the invention features isolated nucleic acids encoding C
s
polypeptides. For example, an isolated nucleic acid may encode a polypeptide comprising a first peptide linked to a second peptide, wherein the first peptide has the sequence Xaa
1
, Ser Xaa
2
Xaa
3
Xaa
4
Asp (SEQ ID NO:1), where Xaa can be any amino acid, and wherein the second peptide is at least 85%, 90%, 95%, 98%, 99% or even 100% identical to a peptide having the amino acid sequence of the predicted human C&agr;1 amino acid sequence beginning at exon 2 (SEQ ID NO:2). This sequence is shown in
FIG. 1A
as part of the full amino acid sequence of the human C&agr;1 polypeptide (SEQ ID NO:4). The amino acid sequence corresponding to SEQ ID NO:2 begins after the “/”. The nucleic acid can also encode the amino acid sequence of SEQ ID NO:2 wherein Xaa
1
is Ala, Xaa
2
is Asn or Ser, Xaa
3
is Ser or Pro, and Xaa
4
is Asn or Ser.
FIG. 1B
shows the full nucleic acid sequence of human C&agr; (SEQ ID NO:5), including the portion beginning at exon 2 (after the “/”) and ending with nucleotide 1133 (SEQ ID NO:3). Both the amino acid and nucleotide sequences of human C&agr; are available from GenBank at Accession No. X07767.
In other embodiments, the second peptide is at least 75% or more, e.g., 100%, identical to the C&agr;1 amino acid sequence beginning at exon 2 of other animals, e.g., mammalian, species of interest, such as dog, cat, horse, cow, or pig. Thus, the C
s
polypeptide can be designed to be species-specific, and can then be used to treat that particular species.
The amino acid sequence of the first peptide can include, e.g., Ala Ser Asn Pro Asn Asp (SEQ ID NO:6), which corresponds to the amino terminal amino acid sequence in mature ovine C
s
, Pro Ser Ser Ser Asn Asp (SEQ ID NO:7), which corresponds to a predicted amino acid sequence encoded by a mouse pseudogene. The amino acid sequence of the first peptide can also include, e.g., Ala Ser Asn Ser Ser Asp (SEQ ID NO:46) or Ala Ser Ser Ser Asn Asp (SEQ ID NO:47). The latter sequences correspond to the amino terminal amino acid sequence in processed human and mouse C
s
, respectively.
The nucleic acid encoding the first peptide can optionally encode the amino acid Met at the amino terminus, i.e., Met Xaa Ser Xaa Xaa Asn Asp (SEQ ID NO:24), Met Ala Ser Asn Pro Asn Asp (SEQ ID NO:8), Met Pro Ser Ser Ser Asn Asp (SEQ ID NO:9), Met Ala Ser Asn Ser Ser Asp (SEQ ID NO:36), or Met Ala Ser Ser Ser Asn Asp (SEQ ID NO:40).
The first peptide can thus be encoded by, e.g, 5′-GCTTCCAACCCCAACGAT-3′ (SEQ ID NO:10), 5′-CCTTCCAGCTCCAATGAT3′ (SEQ ID NO:11), 5′-ATGGCTTCCAACCCCAACGAT-3′ (SEQ ID NO:12), and 5′-ATGCCTTCCAGCTCCAATGAT-3′ (SEQ ID NO:13), 5′-GCTTCCAACTCCAGCGAT-3′ (SEQ ID NO:48), 5′-GCTTCCAGCTC-CAACGAT-3′ (SEQ ID NO:49), 5′-ATGGCTTCCAACTCCAGCGAT-3′ (SEQ ID NO:37), and 5′-ATGGCTTCCAGCTCCAACGAT-3′ (SEQ ID NO:41), which encode the amino acids of SEQ ID NOS: 6-9, 46-47, 36, and 40, respectively.
The isolated nucleic acid can also include a nucleic acid encoding ovine C
s
(SEQ ID NO:14) as shown in
FIGS. 2A-C
. The predicted amino acid sequence of ovine C
s
(SEQ ID NO:15) is also shown in
FIGS. 2A-C
.
In other embodiments, an isolated nucleic acid can encode a polypeptide comprising a first peptide linked to a second peptide, wherein the first peptide has the sequence Met Ala Ser Asn Ser Ser Asp (SEQ ID NO:36) or Ala Ser Asn Ser Ser Asp (SEQ ID NO:46), and the second peptide is at least 85%, 90%, 95%, 98%, 99% or even 100% identical to a peptide having the amino acid sequence of the predicted human C&agr;1 amino acid sequence beginning at exon 2 (SEQ ID NO:2). An example of such a nucleic acid is a nucleic acid encoding the human C
s
polypeptide (SEQ ID NO:34), e.g., the nucleic acid of SEQ ID NO:35.
An “isolated nucleic acid” is a nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. Thus, a recombinant nucleic acid could include some or all of the 5′ non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore i
Leszyk John D.
San Agustin Jovenal T.
Witman George B.
Fish & Richardson P.C.
Monshipouri Maryam
Prouty Rebecca E.
University of Massachusetts
LandOfFree
Sperm associated protein kinase polypeptides, corresponding... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Sperm associated protein kinase polypeptides, corresponding..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Sperm associated protein kinase polypeptides, corresponding... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3056387