Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2002-03-01
2004-10-12
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S029000, C530S300000, C530S350000
Reexamination Certificate
active
06803186
ABSTRACT:
BACKGROUND OF THE INVENTION
In dogs, it is thought that the ability to absorb essential amino acids such as tryptophan and leucine may be limiting to cellular metabolism. Recent research designed to characterize the amino acid absorption capacity of the brush border (lumen facing) membranes of dog enterocytes suggests that peptide absorption may be particularly important given the relatively low amount of free amino acid transport capacity that was observed. Buddington R K, Paulsen D B. Development of the Canine and Feline Gastrointestinal Tract. In: Reinhart G A, Carey D P, eds.
Recent Advances in Canine and Feline Nutrition, Vol. II:
1998
Iams Nutrition Symposium Proceedings
. Wilmington: Orange Frazer Press, 1998; 195-215. Data collected from studies designed to understand the quantitative importance of free versus peptide amino acids in other monogastric animals strongly indicates that peptide-bound amino acids account for the majority of amino acids absorbed by enterocytes from the intestinal lumen (Matthews, D M.
Protein Absorption, Development and Present State of the Subject
, New York: Wiley-Liss, 1991.) and that the rate of peptide-derived amino acid absorption is faster than that by equivalent amounts of free amino acids. Ohkohchi N, Andoh T, Ohi R, Mori S. Defined formula diets alter characteristics of the intestinal transport of amino acid and peptide in growing rats.
J Pediatr Gastroenterol Nutr
1990 May; 10(4):490-6.
Two types of peptide transporters have been cloned from monogastric animals. Liang R, Fei Y J, Prasad P D, Ramamoorthy S, Han H, Yang-Feng T L, Hediger M A, Ganapathy V, Leibach F H. Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.
J Biol Chem
1995 Mar. 24; 270(12):6456-63. Liu W, Liang R, Ramamoorthy S, Fei Y J, Ganapathy M E, Hediger M A, Ganapathy V, Leibach F H. Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney.
Biochim Biophys Acta
1995 May 4; 1235(2):461-6. PepT1 is an H
+
-dependent, low-affinity (mM), high-velocity, transporter that is predominately localized primarily to the brush border membranes of mature enterocytes of intestinal villi. PepT2 is an H
+
-dependent, high-affinity (&mgr;M), low-velocity, transporter that is expressed in the greatest abundance in the apical membranes of renal proximal tubular epithelial cells. An important feature of the peptide transporters is their ability to recognize and transport most di- and tripeptides, albeit with a range of relative affinities for different peptides. In addition, both transporters recognize the &bgr;-lactam antibiotics, and carboxyl-terminal modified free amino acids. The physiologic functions of these transporters are thought to be to absorb di- and tripeptides from the digesta and from the blood, respectively. Although molecular evidence has not been acquired, there is strong biochemical evidence for a different peptide transport protein that functions in the basolateral membrane of these cells. Saito H, Inui K I. Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2
. Am J Physiol
1993 August; 265(2 Pt 1):G289-94. Thwaites D T, Brown C D, Hirst B H, Simmons N L. Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by the expression of H
+
-coupled carriers at both the apical and basal membranes.
J Biol Chem
1993 April 15; 268(11):7640-2.
Research with Caco-2 cells indicates that PepT1 transporter mRNA, protein, and activity increases in a manner consistent with a direct effect of increased extracellular substrate concentrations. Walker D, Thwaites D T, Simmons N L, Gilbert H J, Hirst B H. Substrate upregulation of the human small intestinal peptide transporter, hPepT1.
J Physiol
1998 Mar. 15; 507(Pt 3):697-706. In contrast to mRNAs for essential amino acid transporters, intestinal studies show that the expression of peptide transporter mRNA increases in response to increased dietary protein. Erickson R H, Gum J R Jr, Lindstrom M M, McKean D, Kim Y S. Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.
Biochem Biophys Res Commun
1995 Nov. 2; 216(1):249-57. Similarly, expression in intestinal mucosa of PepT1 mRNA and protein increases in response to tissue trauma, whereas the MRNA for essential amino acid transporters decreases. Tanaka H, Miyamoto K I, Morita K, Haga H, Segawa H, Shiraga T, Fujioka A, Kuoda T, Taketani Y, Hisano S, Fukui Y, Kitagawa K, Takeda E. Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury.
Gastroenterology
1998 April; 114(4):714-23.
Few studies have been conducted to evaluate the potential for the dog to absorb quantitatively significant amounts of essential amino acids in the form of small peptides, and whether this capacity can be regulated by substrate supply. Accordingly, there is still a need to evaluate the potential for the absorption of peptide-bound leucine and tryptophan by putative canine peptide transporters. It would thus be desirable to provide the nucleic acid sequence encoding canine PepT1. It would also be desirable to provide mRNA transcripts corresponding to cPepT1. It would further be desirable to characterize the function of cPepT1 by GlySar uptake and identify di- and tripeptides well recognized by cPepT1, as well as characterize the effect of supplemental peptide substrate on the transport capacity of canine PepT1 (cPepT1).
SUMMARY OF THE INVENTION
The present invention provides novel isolated and purified nucleic acids (RNA or DNA) encoding, or complementary to, canine PepT1 (cPepT1). The nucleic acid may be SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:20 or may be a nucleic acid that hybridizes under moderate or stringent hybridization conditions to any of these sequences. Also provided are peptides encoded by these nucleic acids, such as SEQ ID NO: 13 or SEQ ID NO:21.
The present invention also provides a method for determining canine PepT1-transportability of a peptide, or method for determining a peptide with beneficial nutritional property in an animal, comprising providing an immortalized kidney distal tubule epithelial (Madin-Darby Canine Kidney (MDCK)) cell and a peptide having 2 to 10 amino acids, and determining the amount of the peptide transported into the cell, wherein the amount correlates with the canine PepT1-transportability of the peptide. A peptide with beneficial nutritional properties in an animal is a peptide that contains at least one essential amino acid that is absorbed at a rate higher than the rate of the amino acid if it were free rather than in a peptide-bound form. The peptide may be a dipeptide, tripeptide, or tetrapeptide such as, for example, GlySar, GlyGly, AlaHis, &bgr;-AlaHis (carnosine), GlnGln, GlyMet, LeuMet, LeuTrp, MetLeu, MetMet, MetPhe, MetPro, TrpLeu, TrpTrp, GlnGlu, MetGlu, MetLys, TrpGly, MetGlyMetMet (SEQ ID NO:10), TrpGlyGly, LeuArg, ArgLeu, GlyLeu, or ArgTrp. The cell used in the method may be in medium at a pH of between about 5 and 8; or at a pH of about 5.5 to 7.5, or even at about 6 to 6.5. The peptide may be present at a concentration of about 10 nm to about 50 mM.
The characterization of GlySar uptake by immortalized MDCK cells demonstrates that MDCK cells express PepT1-like activity, confirming detection of PepT1 MRNA expression by MDCK cells and the use of MDCK cells as a model to characterize the biochemical function of canine PepT1.
The cPepT1 of the present invention is also capable of recognizing a variety of di- and tripeptides, including those that contain the essential amino acids leucine and tryptophan, considered to be of especial importance to canine nutrition. In addition, H
+
-dependent peptide transport in cultured MDCK cells can be stimulated by at least two of PepT1 substrates, GlySar and carnosine. Moreover, H
+
-dependent uptake of GlySar by MDCK is sensitive to nutrient deprivation and Insulin-like Growth f
Davenport Gary Mitchell
Matthews Jamie Clyde
Clark Karen F.
Leary Louise N.
McDow-Dunham Kelly L.
The IAMS Company
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