Abstract:
Improved delivery of zinc using haptocorrin or intrinsic factor modified to include a zinc binding sequence that can outcompete dietary zinc inhibitors such as phytin. Known zinc binding sequences can be assayed to determine competitiveness with respect to phytin and, if successful, incorporated into the B 12  binding site of haptocorrin or intrinsic factor. Thus a method of producing functional human haptocorrin or intrinsic factor which binds to zinc includes the steps of modifying human TCN1 or GIF to comprise one or more zinc-binding site sequences, constructing a vector containing the modified human TCN1 or GIF sequence, and introducing the vector into a host cell for a time and under conditions sufficient for expression of the functional human haptocorrin or intrinsic factor. The resulting zinc binding complex can be orally administered to zinc deficient individuals to improve the amount of dietary zinc available to the individuals.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application Ser. No. 61/823,594, filed on May 15, 2013, and entitled “Zinc Delivery System Using Intrinsic Factor or Haptocorrin,” the entire disclosure of which is incorporated herein by reference. 
    
    
     BACKGROUND 
     The present invention relates to methods and systems for oral delivery of zinc and, more specifically, to the use of intrinsic factor or haptocorrin to enhance oral zinc delivery and zinc absorption. 
     Zinc deficiency is widespread and affects the health and well-being of populations worldwide. As much as 25% of the world&#39;s population may have inadequate levels of zinc in their diet due to a combination of factors including limited access to zinc-rich foods such as animal products, oysters, or shellfish, and the abundance of zinc inhibitors such as phytin that are common in plant-based foods. Thus, even if an individual&#39;s zinc intake levels are adequate, the levels of inhibitors in the diet through consumption of foods such as cereals, corn, and rice may mean that inadequate amounts of zinc are absorbed. As a result, zinc deficiency may not necessarily be treated by providing dietary zinc supplements or increasing the amount of zinc rich foods consumed by individuals having diets comprised of foods containing zinc inhibitors. 
     A human being with adequate levels of zinc comprises 2-4 grams of the mineral dispersed throughout their body, with major concentrations found in the brain, muscles, and bones. Zinc is utilized by the human body in a wide array of different metabolic processes, including the metabolism of RNA and DNA, signal transduction, gene expression, apoptosis, and, perhaps most importantly, the structure and function of proteins such as enzymes. 
     Insufficient levels of zinc in the body, however, can have devastating effects ranging from minor to devastating. Minor side-effects of low zinc levels include diarrhea, acne, and low testosterone, while major issues include cognitive and motor function impairment, chronic renal disease, and the malfunction of processes such as eyesight, memory, and smell, among many others. As a result, zinc deficiency can result in significant reduction of quality of life, leading even to increased mortality rates. 
     Accordingly, there is a need in the art for methods and systems that facilitate zinc supplementation in a manner that avoids dietary zinc inhibitors and provides an adequate amount of zinc to be absorbed by a zinc-deficient individual. 
     BRIEF SUMMARY 
     The present invention is directed to methods and systems to enhance oral zinc delivery and zinc absorption using intrinsic factor or haptocorrin. In view of the foregoing, embodiments are directed to a system of protecting orally delivered zinc from degradation using intrinsic factor bound to the zinc. Known zinc binding sequences are tested for competitiveness against phytin, and successful sequences are incorporated into haptocorrin or intrinsic factor, such as in a B 12  binding location since the ability to bind B 12  is not a priority. The resulting zinc binding complex may then be orally administered to zinc deficient populations to increase the amount of available supplemental or dietary zinc. 
     Generally, in one aspect, a nucleic acid molecule comprises a chimeric gene derived from human haptocorrin comprising a first sequence encoding a first region of haptocorrin, a second sequence encoding a zinc-binding site, and a third sequence encoding a second region of haptocorrin. 
     In some embodiments, the molecule is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 4. 
     In some embodiments, the zinc-binding site is inserted within an α domain of the human haptocorrin. 
     In some embodiments, the zinc-binding site is inserted between an α domain and a β domain of the human haptocorrin. 
     Generally, in one aspect, a polypeptide comprises a chimeric protein derived from human haptocorrin, the polypeptide comprising a zinc-binding site located between a first region of haptocorrin and a second region of haptocorrin. 
     In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 6. 
     In some embodiments, the polypeptide is capable of reversibly binding zinc. 
     Generally, in one aspect, a nucleic acid molecule comprises a chimeric gene derived from human intrinsic factor comprising a first sequence encoding a first region of intrinsic factor, a second sequence encoding a zinc-binding site, and a third sequence encoding a second region of intrinsic factor. 
     Generally, in one aspect, a polypeptide comprises a chimeric protein derived from human intrinsic factor, the polypeptide comprising a zinc-binding site located between a first region of intrinsic factor and a second region of intrinsic factor. 
     Generally, in one aspect, a method of producing functional human haptocorrin which binds to zinc includes the steps of: modifying human TCN1 to comprise one or more zinc-binding site sequences; constructing a vector comprising the modified human TCN1; and introducing the vector into a host cell for a time and under conditions sufficient for expression of the functional human haptocorrin. 
     In some embodiments, the modified human TCN1 is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 4. 
     In some embodiments, the functional human haptocorrin comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 6. 
     In some embodiments, the method further includes the step of isolating the expressed functional human haptocorrin. 
     In some embodiments, the method further includes the step of incubating the isolated human haptocorrin with zinc. 
     Generally, in one aspect, a method of producing functional human intrinsic factor which binds to zinc includes the steps of: modifying human GIF to comprise one or more zinc-binding site sequences; constructing a vector comprising the modified human GIF; and introducing the vector into a host cell for a time and under conditions sufficient for expression of the functional human intrinsic factor. 
     In some embodiments, the method further includes the step of isolating the expressed functional human intrinsic factor. 
     In some embodiments, the method further includes the step of incubating the isolated human intrinsic factor with zinc. 
     Generally, in one aspect, a method for testing the zinc binding affinity of a modified human haptocorrin, the method inclusion the steps of: modifying human TCN1 to comprise one or more zinc-binding site sequences; constructing a vector comprising the modified human TCN1; introducing the vector into a host cell for a time and under conditions sufficient for expression of the functional human haptocorrin; isolating the expressed functional human haptocorrin; incubating the isolated human haptocorrin with zinc; exposing the zinc-bound haptocorrin to a zinc-binding compound; and determining the effect of exposure to the zinc-binding compound on the exposed haptocorrin. 
     In some embodiments, the zinc-binding compound is a phytate. 
     In some embodiments, the exposing step is performed under conditions comprising a pH of 3 or lower. 
     Generally, in one aspect, a method of binding and releasing zinc using a chimeric protein includes the steps of: providing a chimeric protein derived from human haptocorrin, the chimeric protein comprising a zinc-binding site located between a first region of haptocorrin and a second region of haptocorrin; binding the polypeptide to zinc; and administering the zinc-bound polypeptide orally. 
     In some embodiments, the chimeric protein is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 6. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
       The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which: 
         FIG. 1  is a schematic of intrinsic factor modified for the delivery of zinc according to the present invention; 
         FIG. 2  is a schematic of haptocorrin modified for the delivery of zinc according to the present invention; 
         FIG. 3  is a graph of eluent from a B 12  column on which cell extract containing an expressed modified haptocorrin protein was loaded; and 
         FIG. 4  is an image of eluent from the column of  FIG. 3  testing positive for haptocorring using a haptocorrin primary antibody. 
     
    
    
     DETAILED DESCRIPTION 
     It is often desirable to provide zinc supplements to zinc-deficient individuals. Prevalent zinc deficiency affects the health and well-being of millions of people around the world. Not only do people have inadequate levels of zinc in their diet due to limited access to zinc-rich foods, but the abundance of zinc inhibitors common in plant-based foods block ingested zinc. As a result, zinc deficiency may not necessarily be treated by providing dietary zinc supplements or increasing the amount of zinc rich foods consumed by individuals having diets comprised of foods containing zinc inhibitors. 
     In light of pervasive zinc deficiency caused by both limited access to zinc-rich foods and the abundance of zinc inhibitors, Applicants have recognized that it is desirable to provide zinc supplementation to zinc-deficient individuals in a manner that avoids dietary zinc inhibitors. 
     In view of the foregoing, various embodiments are directed to methods and systems to enhance oral zinc delivery and zinc absorption using intrinsic factor or haptocorrin. In particular, orally delivered zinc is protected from degradation using intrinsic factor bound to the zinc. For example, known zinc binding sequences are tested for competitiveness against phytin, and successful sequences are incorporated into haptocorrin or intrinsic factor, such as in a B12 binding location since the ability to bind B12 is not a priority. The resulting zinc binding complex may then be orally administered to zinc deficient populations to increase the amount of available supplemental or dietary zinc. 
     Modifying Intrinsic Factor to Bind Zinc 
     According to one embodiment, the structure of intrinsic factor (“IF”) is modified to include a zinc binding sequence that will capture zinc and protect it from complexation with dietary phytates and other zinc inhibitors. Un-modified IF is a 43,420 Da glycoprotein produced from the GIF gene, and consisting of approximately 399 amino acids with an additional 17 of those amino acids at the N-terminus acting as a secretory signal sequence that is cleaved during processing. The structure of human IF (“hIF”) is composed of α and β heterodomains, where the α domain consists of approximately 270 residues (7-273) and the β domain consists of approximately 110 residues (274-399). The α domain is composed of an α 6 /α 6  helical barrel, while the β domain consists of β strands. 
     The one or more zinc binding sequences in the modified IF will capture zinc and then protect that bound zinc from other agents such as dietary phytates and other zinc inhibitors. The zinc-bound IF will travel substantially unaffected through the stomach and into the small intestine. Once in the small intestine, the complex will bind to the IF receptor expressed on epithelial cells of the ileum wall. The activated IF receptor complex then undergoes endocytosis, releasing the zinc into the blood serum. Accordingly, it is important that the modified IF not only be able to bind zinc, but must also bind and activate the IF receptor in the ileum. 
     Referring to  FIG. 1 , an embodiment of the present invention comprises a modification to IF to replace or augment at least a portion of the interface region between the a and  13  heterodomains with a zinc binding sequence. More particularly, an embodiment of the present invention comprises replacement of one or more amino acids that lie closest to the interface between the α domain (residues 7-273) and the β domain (residues 274-399), and preferably just within the beginning of the β domain. This region of IF is responsible for B 12  binding, which is not necessary for the present invention. 
     Modifying Haptocorrin to Bind Zinc 
     According to another embodiment, the structure of haptocorrin is modified to include a zinc binding sequence that will capture zinc and protect it from degradation. Unmodified haptocorrin is one of three B 12  transport proteins and is a 47 kDa, highly glycosolated, protein responsible for initial B 12  binding and transport from the mouth, through the stomach, to the intestine. Once in the intestine, haptocorrin is degraded due to an increase in pH and enzymatic digestion by trypsin and chymotrypsin, and the bound B 12  is thus released. Haptocorrin is also present in the blood serum and is hypothesized to remove B 12  analogs that are not able to successfully undergo the body&#39;s metabolic pathways. Haptocorrin includes an α domain comprised of a helical barrel (residues 1-287) and a β domain comprised of strands (residues 309-410) that are interconnected by a flexible linker. 
     EXAMPLE 1 
     Modified Haptocorrin Sequence 
     Referring to  FIG. 2 , another embodiment of the present invention comprises the modification of haptocorrin to replace at least a portion of the α-β interface region between the a domain (residues 1-287) and the β domain (309-410) with a zinc binding sequence. As with IF, this portion of haptocorrin is responsible for B 12  binding, which is not necessary for the present invention. 
     EXAMPLE 2 
     Selection of Zinc Binding Sequences 
     Traditional zinc-binding domains that rely on histidine and cysteine for metal coordination, such as two invariant pairs of cysteine and histidine that coordinate a zinc atom, are not efficient below a pH of 3. As a result, in accordance with an embodiment the present invention relies on polycarboxylate coordination using the side chains of Asp and Glu. Polyaspartate tags are well-established effective metal chelators and will likely provide enough stability, even at a low pH. Specifically, in accordance with one embodiment, the present invention encompasses the following sequences:
 
 G/A −( XG ) n  
 
 G −(( X 3) G ( X 3)) m−G  
 
 Xo  
 
where X=Glu or Asp; n=4-7; m=2-3; and o=6-10, although many other variations and zinc-binding sequences are possible.
 
     Zinc binding sequences may be evaluated to determine their binding affinities (Kd) to zinc, particularly with regard to the binding affinity of phytin to zinc. Sequences having a sufficiently high or higher affinity for zinc than phytin may be inserted into the predetermined location of haptocorrin or IF, and then subjected to a phytin challenge to determine whether the test complex can outcompete phytin for zinc. This challenge can be performed in vitro and is preferably done at a pH of 2-3 to replicate the acid environment of the intestines. 
     EXAMPLE 3 
     Location of Zinc Binding Sequences 
     Selecting the location within haptocorrin and intrinsic factor to insert the one or more zinc-binding sequences is also vital to the successful structure and function of the modified protein, including both zinc-binding and protection of the zinc as the protein travels through the stomach and into the small intestine. The selected location must result in a stable protein that can bind zinc and protect it from the environment of the stomach. 
     Preferably, for modified haptocorin for example, the selected location should result in a stable protein that is also capable of binding vitamin B 12  in addition to the zinc. For example, the modified haptocorrin can bind the zinc in the bottom of the vitamin B 12  pocket and then the vitamin B 12  can subsequently bind within the pocket, providing additional protection. Haptocorrin consists of an α domain comprised of a helical barrel (residues 1-287) and a β domain comprised of β strands (residues 309-410) that are interconnected by a flexible linker. According to one embodiment, the one or more zinc binding site sequences are inserted within the haptocorrin gene such that the binding site is at the bottom of the B 12  binding site, located at the bottom of the α domain, where the zinc will be highly protected. That location will also avoid disruption of B 12  binding, allowing B 12  to bind to the pocket on top of the zinc which provides further protection. According to another embodiment, the one or more zinc binding site sequences are inserted within the haptocorrin gene such that the binding site is at the loop between the α domain and a β domain. 
     EXAMPLE 4 
     Modified Haptocorrin Sequence 1 (YNHCZn) 
     As described herein, and in accordance with an embodiment, the human haptocorrin protein encoded by the TCN1 gene can be modified to include one or more zinc-binding sites. For example, SEQ ID NO: 1 is an example of the modified TCN1 gene containing the zinc-binding site sequence RDADADR (SEQ ID NO: 2) (a common zinc-binding site sequence), and which results in expression of the modified human haptocorrin protein identified by SEQ ID NO: 3. According to this embodiment, the 21-nucleotide sequence for the zinc-binding “RDADADR” sequence is inserted between nucleotides 423 and 424 of human haptocorrin. 
     According to one embodiment, to test the expression of modified haptocorrin sequence 1 (YNHCZn), the modified gene (e.g., SEQ ID NO: 1) was expressed in insect cells The cell extract (containing the modified haptocorrin) was loaded onto a B 12  column and then eluted (shown in  FIG. 3 ). The eluent was then tested for the presence of haptocorrin using a haptocorrin primary antibody, and positive results (see  FIG. 4 ) indicated that the insect cell expressed modified haptocorrin sequence 1 (YNHCZn) is indeed expressed, and that the modified protein is capable of binding B 12 . 
     EXAMPLE 5 
     Modified Haptocorrin Sequence 2 (HCZn2b) 
     As described herein, and in accordance with an embodiment, the human haptocorrin protein encoded by the TCN1 gene can be modified to include one or more zinc-binding sites. Another embodiment of a modified TCN1 gene containing a zinc-binding site sequence is SEQ ID NO: 4, which contains the zinc-binding site sequence MTSTTLVKCACEPCLCNVDPSKAIDRNGLYYCSEACADGHTGGSKGCGHTGCNCHG (SEQ ID NO: 5) which is a SmtA metallothionein sequence. Metallothioneins are small, cysteine-rich proteins that are extremely good at binding metal ions. For example, the SmtA gene from  Synechococcus  binds zinc ions extremely well. According to this embodiment, the 168-nucleotide sequence encoding for this zinc-binding sequence is inserted between nucleotides 426 and 427 of human haptocorrin, and results in expression of the modified human haptocorrin protein identified by SEQ ID NO: 6. 
     EXAMPLE 6 
     Testing of Efficacy of Zinc Binding Complex 
     Once zinc binding sequences that can outcompete phytin are selected, those sequences can be incorporated into IF or haptocorrin, so that the resulting zinc binding complex may be tested for efficacy in humans using animal models. For example, the zinc binding complex may be orally administered along with zinc and phytin to a test group, and just the zinc and phytin delivered orally to a control group. Blood may then be drawn from both groups over a predetermined period of time to determine whether the administration of the zinc binding complex improves uptake of zinc in the presence of phytin. 
     Although the present invention has been described in connection with a preferred embodiment, it should be understood that modifications, alterations, and additions can be made to the invention without departing from the scope of the invention as defined by the claims.