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Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

Lacroix IM, Li-Chan EC - Int J Mol Sci (2014)

Bottom Line: Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied.While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme.This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Land and Food Systems, Food, Nutrition and Health Program, the University of British Columbia, 2205 East Mall, Vancouver, BC V6T 1Z4, Canada. isablac@mail.ubc.ca.

ABSTRACT
The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αK(i) = 76 µM), 105LAHKALCSEK114 (K(i) = 217 µM) and 110LCSEKLDQWL119 (K(i) = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

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Related in: MedlinePlus

Schematic representation of peptide array synthesis, binding and inhibition experiments. Binding of α-lactalbumin-derived decamers to the dipeptidyl-peptidase IV (DPP-IV) enzyme was investigated directly on the cellulose membrane, whereas the inhibition of DPP-IV was measured using a colorimetric assay with the cleaved peptides.
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ijms-15-20846-f001: Schematic representation of peptide array synthesis, binding and inhibition experiments. Binding of α-lactalbumin-derived decamers to the dipeptidyl-peptidase IV (DPP-IV) enzyme was investigated directly on the cellulose membrane, whereas the inhibition of DPP-IV was measured using a colorimetric assay with the cleaved peptides.

Mentions: The interaction between the DPP-IV enzyme and deca-peptides spanning the entire α-lactalbumin sequence (Table S1) was first determined by immunoassay and visualized using an enhanced chemiluminescence substrate (Figure 1). As shown in Figure 2, the probing of the peptide array with DPP-IV revealed that a number of α-lactalbumin-derived peptides are able to interact with the enzyme (dark spots on the array). Since every consecutive spot on the membrane differs by only one amino acid, the presence of consecutive dark spots indicates that some regions of the α-lactalbumin molecule such as 1EQLTKCEVFRELK13 (spots A1–A4), 45NDSTEYGLFQINNKIWCK62 (spots E1–E9) and 89IMCVKKILDKVGINYWLAHKALCSEKL115 (spots I1–J7) were able to bind to DPP-IV while others like 61CKDDQNPHSSNICN74 (spots F6–F10) and 68HSSNICNISCDKFLD82 (spots G2–G7) did not seem to interact with the enzyme.


Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

Lacroix IM, Li-Chan EC - Int J Mol Sci (2014)

Schematic representation of peptide array synthesis, binding and inhibition experiments. Binding of α-lactalbumin-derived decamers to the dipeptidyl-peptidase IV (DPP-IV) enzyme was investigated directly on the cellulose membrane, whereas the inhibition of DPP-IV was measured using a colorimetric assay with the cleaved peptides.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264199&req=5

ijms-15-20846-f001: Schematic representation of peptide array synthesis, binding and inhibition experiments. Binding of α-lactalbumin-derived decamers to the dipeptidyl-peptidase IV (DPP-IV) enzyme was investigated directly on the cellulose membrane, whereas the inhibition of DPP-IV was measured using a colorimetric assay with the cleaved peptides.
Mentions: The interaction between the DPP-IV enzyme and deca-peptides spanning the entire α-lactalbumin sequence (Table S1) was first determined by immunoassay and visualized using an enhanced chemiluminescence substrate (Figure 1). As shown in Figure 2, the probing of the peptide array with DPP-IV revealed that a number of α-lactalbumin-derived peptides are able to interact with the enzyme (dark spots on the array). Since every consecutive spot on the membrane differs by only one amino acid, the presence of consecutive dark spots indicates that some regions of the α-lactalbumin molecule such as 1EQLTKCEVFRELK13 (spots A1–A4), 45NDSTEYGLFQINNKIWCK62 (spots E1–E9) and 89IMCVKKILDKVGINYWLAHKALCSEKL115 (spots I1–J7) were able to bind to DPP-IV while others like 61CKDDQNPHSSNICN74 (spots F6–F10) and 68HSSNICNISCDKFLD82 (spots G2–G7) did not seem to interact with the enzyme.

Bottom Line: Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied.While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme.This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Land and Food Systems, Food, Nutrition and Health Program, the University of British Columbia, 2205 East Mall, Vancouver, BC V6T 1Z4, Canada. isablac@mail.ubc.ca.

ABSTRACT
The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αK(i) = 76 µM), 105LAHKALCSEK114 (K(i) = 217 µM) and 110LCSEKLDQWL119 (K(i) = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

Show MeSH
Related in: MedlinePlus