NZ615495B2 - Anti-inflammatory proteins and peptides and methods of preparation and use thereof - Google Patents
Anti-inflammatory proteins and peptides and methods of preparation and use thereof Download PDFInfo
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- NZ615495B2 NZ615495B2 NZ615495A NZ61549513A NZ615495B2 NZ 615495 B2 NZ615495 B2 NZ 615495B2 NZ 615495 A NZ615495 A NZ 615495A NZ 61549513 A NZ61549513 A NZ 61549513A NZ 615495 B2 NZ615495 B2 NZ 615495B2
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Abstract
Discloses the use of a functional fragment of a major royal jelly protein (MRJP) for preparation of a medicament for reducing inflammation in a cellular tissue in a patient; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises 2 to 20 amino acids of the last 20 amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO). agment comprises 2 to 20 amino acids of the last 20 amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
Description
ANTI-INFLAMMATORY PROTEINS AND PEPTIDES AND METHODS OF
PREPARATION AND USE THEREOF
RELATED APPLICATION
This application claims the benefit of New Zealand provisional
application NZ 600847 filed 22 June 2012, the entirety of which is incorporated herein
by reference.
FIELD OF THE INVENTION
The present disclosure relates to anti-inflammatory proteins and
peptides, their uses, and methods of their detection.
BACKGROUND OF THE INVENTION
Honey has been used for centuries by cultures through the world for its
multiple health benefits. Two of the most important health benefits of honey are its
anti-bacterial and anti-inflammatory properties. Manuka honey, which is produced by
bees that collect nectar from Leptospermum scoparium, a plant native to New Zealand
and southern Australia, has been identified as being a variety of honey that exhibits
particularly effective anti-bacterial and anti-inflammatory properties. Jelly Bush honey,
which is produced by bees that collect nectar from Leptospermum polygalifolium, a
plant native to Australia, has also been identified as being a variety of honey that
exhibits particularly effective anti-bacterial and anti-inflammatory properties.
[0004] Recently, it was discovered that the chemical, methylglyoxal (MGO,
also called 2-oxopropanal and pyruvaldehyde), is a major component of the anti-
bacterial activity of Leptospermum derived honey, such as manuka honey and jelly
bush honey. Manuka honey and jelly bush honey samples contain greater
concentrations of MGO, and have a higher amount of anti-bacterial activity as
compared to honey samples with lower concentrations of MGO. MGO is believed to
confer antibacterial properties on honey because MGO is a highly chemically reactive
compound, and MGO can readily react with cellular molecules. The chemical reactions
between MGO and cellular molecules in bacteria damage bacterial molecules that are
important for viability. In this way, MGO functions as an antibacterial agent.
The presence of high levels of MGO in the honey is a feature that
distinguishes manuka honey and jelly bush honey from other varieties of honey. While
most varieties of honey exhibit some anti-bacterial activity, the anti-bacterial activity in
most varieties of honey is primarily a result of the presence of hydrogen peroxide in the
honey. Leptospermum derived honey, in contrast, exhibits anti-bacterial activity
primarily because of the presence of MGO in the honey.
In 2004, Kohno et. al. examined the anti-inflammatory effects or actions
of royal jelly at a cytokine level. The results suggest that royal jelly has anti-
inflammatory actions brought about by an inhibition of pro-inflammatory cytokine
production, such as TNF- IL-6 and IL-1, by activated macrophages. The study
further suggests that the active fractions or components from the royal jelly extracts are
sized between 5 kDa and 30 kDa. Thus, most honeys may have a weak anti-
inflammatory effect due to royal jelly proteins that occur in the honey.
While multiple mechanisms of action of the anti-bacterial activity of
manuka honey have been elucidated, the mechanisms whereby manuka honey functions
as an anti-inflammatory agent have remained unknown. There is a need to develop
anti-inflammatory agents based on honey, as many anti-inflammatory agents currently
available have major drawbacks to their use. For example, COX-2 inhibitors, a form of
non-steroidal anti-inflammatory drug (NSAID), may increase the risk of heart attack
and stroke in patients, and aspirin may increase the risk of gastrointestinal bleeding.
Additionally, corticosteroids are reported to inhibit the growth of epithelial cells and
NSAIDs are reported as being cytotoxic so both of these classes of anti-inflammatory
agents are unsuitable for use in wound care. Anti-inflammatory agents derived from
honey may have fewer toxic side effects in one or more areas than drugs currently
available, and may also offer different possible uses than anti-inflammatory drugs
currently available.
Described in the co-pending application PCT/NZ/2011/000271 is a
modified apalbumin of approximately 55 – 75 kDa from manuka honey that results
from the high levels of methylglyoxal found in manuka honey. The inventors have
identified that the modified apalbumin or major royal jelly protein has significantly
greater anti-inflammatory properties than an unmodified apalbumin or major royal jelly
protein.
SUMMARY OF THE INVENTION
Described herein are apalbumins, also known as major royal jelly
proteins, and functional fragments thereof, which have at least one lysine or arginine
amino acid chemically modified by methylglyoxal (MGO), and which exhibit enhanced
anti-inflammatory effects.
In a first aspect the present invention provides a method of reducing
inflammation in a cellular tissue, comprising the step of contacting the cellular tissue
with a functional fragment of a major royal jelly protein (MRJP); wherein the
functional fragment has been isolated, enriched, synthesized, or recombinantly
produced; wherein the functional fragment comprises 2 to 20 amino acids of the last 20
amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue
of the functional fragment has been chemically modified by methylglyoxal (MGO). In
one embodiment the major royal jelly protein (MRJP) is selected from the group
consisting of MRJP1 (SEQ ID NO: 1), MRJP2 (SEQ ID NO: 2), MRJP3 (SEQ ID NO:
3), MRJP4 (SEQ ID NO: 4), MRJP5 (SEQ ID NO: 5), MRJP6 (SEQ ID NO: 6),
MRJP7 (SEQ ID NO: 7), MRJP8 (SEQ ID NO: 8), and MRJP9 (SEQ ID NO: 9).
In another aspect the present invention provides a method of reducing
inflammation in a cellular tissue, comprising the step of contacting the cellular tissue
with a functional fragment of a major royal jelly protein (MRJP); wherein the
functional fragment has been isolated, enriched, synthesized, or recombinantly
produced; wherein the functional fragment comprises an amino acid sequence selected
from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR
(SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK
(SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the
functional fragment has been chemically modified by methylglyoxal (MGO). In one
embodiment the inflammation is associated with one or more of the group consisting
of: an inflammatory disorder, a cardiovascular disorder, a neurological disorder, a
pulmonary disorder, a proliferative disorder, an infectious disease or associated
syndrome, an allergic, immunological or autoimmune disorder, and inflammation
associated with a wound.
In a further aspect there is provided a method of inhibiting Cathepsin B
activity in a cellular tissue, comprising the step of contacting the cellular tissue with a
functional fragment of a major royal jelly protein (MRJP); wherein the functional
fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein
the functional fragment comprises an amino acid sequence selected from the group
consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO:
127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO:
137); and wherein a lysine or arginine amino acid residue of the functional fragment
has been chemically modified by methylglyoxal (MGO).
In a further aspect, there is provided an isolated functional fragment of a
major royal jelly protein (MRJP), wherein the functional fragment comprises an amino
acid sequence selected from the group consisting of:
i. KISIHL (SEQ ID NO: 10);
ii. KNNNQNDN (SEQ ID NO: 11);
iii. KLH (SEQ ID NO: 12);
iv. KSNNRHNNND (SEQ ID NO: 13);
v. KHNN (SEQ ID NO: 14);
vi. KNQAHLD (SEQ ID NO: 15);
vii. KNTRCISP (SEQ ID NO: 16);
viii. KTNFFSIFL (SEQ ID NO: 17); and
wherein a lysine amino acid residue of the functional fragment has been chemically
modified by methylglyoxal (MGO).
In another aspect the present invention provides an isolated functional
fragment of a major royal jelly protein (MRJP); wherein the functional fragment
comprises an amino acid sequence selected from the group consisting of: LVK (SEQ
ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO:
128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine
or arginine amino acid residue of the functional fragment has been chemically modified
by methylglyoxal (MGO).
In another aspect there is provided a method of producing an anti-
inflammatory molecule that is an apalbumin protein or functional fragment thereof by
modifying royal jelly, the method including the step of reacting royal jelly with at least
0.1% MGO at between 18 and 37 degrees Celsius.
[0016] In another aspect there is provided a method of enriching the anti-
inflammatory molecules in a Leptospermum genus derived MGO containing honey
comprising the step of adding Major Royal Jelly Protein to the honey.
In a further aspect there is provided a method of identifying (i) the anti-
inflammatory capacity or (ii) MGO-modified major royal jelly protein concentration of
a sample of honey, comprising the step of: assaying the Cathepsin B inhibition levels
of the honey sample.
In another aspect there is provided a method of inhibiting Cathepsin B
activity in a cellular tissue, comprising the step of contacting the cellular tissue with a
functional fragment of a major royal jelly protein (MRJP); wherein the functional
fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein
the functional fragment comprises 2 to 20 amino acids of the last 20 amino acids at the
C-terminus of the protein; and wherein a lysine amino acid residue of the functional
fragment has been chemically modified by methylglyoxal (MGO). In one embodiment
the major royal jelly protein (MRJP) is selected from the group consisting of MRJP1
(SEQ ID NO: 1), MRJP2 (SEQ ID NO: 2), MRJP3 (SEQ ID NO: 3), MRJP4 (SEQ ID
NO: 4), MRJP5 (SEQ ID NO: 5), MRJP6 (SEQ ID NO: 6), MRJP7 (SEQ ID NO: 7),
MRJP8 (SEQ ID NO: 8), and MRJP9 (SEQ ID NO: 9).
The foregoing brief summary broadly describes the features and
technical advantages of certain embodiments of the present invention. Further
technical advantages will be described in the detailed description of the invention and
Examples that follows. Novel features that are believed to be characteristic of the
invention will be better understood from the detailed description of the invention when
considered in connection with any accompanying figures and examples. However, the
Figures and Examples provided herein are intended to help illustrate the invention or
assist with developing an understanding of the invention, and are not intended to limit
the invention's scope.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1B: SDS PAGE analysis of MRJP. Figure 1A: Bolt® 4-
12% Bis-Tris Plus gel. Lane 1: Protein marker (SeeBlue Plus2 Pre-stained Standard.
Lane 2: Crude royal jelly is dissolved in double distilled water (10 µl load of
approximately 5 mg/mL crude). Lane 3: Crude royal jelly is dissolved in double
distilled water (10 µl load of approximately 2 mg/mL crude). Figure 1B: NuPAGE®
Novex® 4-12% Bis-Tris gel. Lane 3: Crude royal jelly dissolved in double distilled
water (soluble fractions, pH 4). Lane 4: Fraction 2 of 5 mL HiTrap™ de-salt column in
6 M urea containing no PMSF, pH 4, 48 h at room temperature. Lane 5: Fraction 2 of 5
mL HiTrap™ de-salt column in 6 M urea containing 1 mM PMSF, pH 4, 48 h at room
temperature. Lane 6: Fraction 2 of 5 mL HiTrap™ de-salt column in 6 M urea
containing no PMSF, pH 8, 48 h at room temperature. Lane 7: Fraction 2 of 5 mL
HiTrap™ de-salt column in 6 M urea containing 1 mM PMSF, pH 8, 48 h at room
temperature. Lane 8: Fraction 2 of 5 mL HiTrap™ de-salt column in 6 M urea
containing no PMSF, pH 8, 168 h at room temperature. Lane 9: Fraction 2 of 5 mL
HiTrap™ de-salt column in 6 M urea containing 1 mM PMSF, pH 8, 168 h at room
temperature. Lane 10: Protein marker.
Figure 2: Native PAGE Novex® 4-16% Bris-Tri gel of MGO-
modified MRJP at pH 4.0 and 7.0, day 1 and day 2. Lane 1: Protein Standard contains
protein bands 20-12,000 kDa]. Lane 2: MGO alone in PBS buffer, pH 7.4 after 24 h.
Lane 3: MGO alone in 0.2 M sodium acetate buffer, pH 4.0 after 24 h. Lane 4: MRJP
in PBS buffer, pH 7.4 after 24 h. Lane 5: MRJP in 0.2 M sodium acetate buffer, pH 4.0
after 24 h. Lane 6: MRJP reacted with MGO in PBS buffer, pH 7.4 after 24 h. Lane 7:
MRJP reacted with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 24 h. Lane 8:
MGO alone in PBS buffer, pH 7.4 after 48 h. Lane 9: MGO alone in 0.2 M sodium
acetate buffer, pH 4.0 after 48 h. Lane 10: MRJP in PBS buffer, pH 7.4 after 48 h.
Lane 11: MRJP in 0.2 M sodium acetate buffer, pH 4.0 after 48 h. Lane 12: MRJP
reacted with MGO in PBS buffer, pH 7.4 after 48 h. Lane 13: MRJP reacted with
MGO in 0.2 M sodium acetate buffer, pH 4.0 after 48 h. Lane 14: BSA control. Lane
15: Protein marker (same as Lane 1).
Figure 3: Native PAGE Novex® 4-16% Bris-Tri gel of MGO-
modified MRJP at pH 4.0 and 7.0, day 3 and day 4. Lane 1: Protein Standard contains
protein bands 20-12,000 kDa. Lane 2: MGO alone in PBS buffer, pH 7.4 after 72 h.
Lane 3: MGO alone in 0.2 M sodium acetate buffer, pH 4.0 after 72 h. Lane 4: MRJP
in PBS buffer, pH 7.4 after 72 h. Lane 5: MRJP in 0.2 M sodium acetate buffer, pH 4.0
after 72 h. Lane 6: MRJP reacted with MGO in PBS buffer, pH 7.4 after 72 h. Lane 7:
MRJP reacted with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 72 h. Lane 8:
MGO alone in PBS buffer, pH 7.4 after 96 h. Lane 9: MGO alone in 0.2 M sodium
acetate buffer, pH 4.0 after 96 h. Lane 10: MRJP in PBS buffer, pH 7.4 after 96 h.
Lane 11: MRJP in 0.2 M sodium acetate buffer, pH 4.0 after 96 h. Lane 12: MRJP
reacted with MGO in PBS buffer, pH 7.4 after 96 h. Lane 13: MRJP reacted with
MGO in 0.2 M sodium acetate buffer, pH 4.0 after 96 h. Lane 14: BSA control. Lane
: Protein marker (same as Lane 1).
Figure 4: Native PAGE Novex® 4-16% Bris-Tri gel of MGO-
modified MRJP at pH 4.0 and 7.0, day 5 and day 6. Lane 1: Protein Standard contains
protein bands 20-12,000 kDa. Lane 2: MGO alone in PBS buffer, pH 7.4 after 120 h.
Lane 3: MGO alone in 0.2 M sodium acetate buffer, pH 4.0 after 120 h. Lane 4: MRJP
in PBS buffer, pH 7.4 after 120 h. Lane 5: MRJP in 0.2 M sodium acetate buffer, pH
4.0 after 120 h. Lane 6: MRJP reacted with MGO in PBS buffer, pH 7.4 after 120 h.
Lane 7: MRJP reacted with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 120 h.
Lane 8: MGO alone in PBS buffer, pH 7.4 after 144 h. Lane 9: MGO alone in 0.2 M
sodium acetate buffer, pH 4.0 after 144 h. Lane 10: MRJP in PBS buffer, pH 7.4 after
144 h. Lane 11: MRJP in 0.2 M sodium acetate buffer, pH 4.0 after 144 h. Lane 12:
MRJP reacted with MGO in PBS buffer, pH 7.4 after 144 h. Lane 13: MRJP reacted
with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 144 h. Lane 14: BSA control.
Lane 15: Manuka honey (crude)
[0024] Figure 5: Native PAGE Novex® 4-16% Bris-Tri gel of MGO-
modified MRJP at pH 4.0 and 7.0, day 7 and day 9. Lane 1: Protein Standard contains
protein bands 20-12,000 kDa. Lane 2: MGO alone in PBS buffer, pH 7.4 after 168 h.
Lane 3: MGO alone in 0.2 M sodium acetate buffer, pH 4.0 after 168 h. Lane 4: MRJP
in PBS buffer, pH 7.4 after 168 h. Lane 5: MRJP in 0.2 M sodium acetate buffer, pH
4.0 after 168 h. Lane 6: MRJP reacted with MGO in PBS buffer, pH 7.4 after 168 h.
Lane 7: MRJP reacted with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 168 h.
Lane 8: MGO alone in PBS buffer, pH 7.4 after 216 h. Lane 9: MGO alone in 0.2 M
sodium acetate buffer, pH 4.0 after 216 h. Lane 10: MRJP in PBS buffer, pH 7.4 after
216 h. Lane 11: MRJP in 0.2 M sodium acetate buffer, pH 4.0 after 216 h. Lane 12:
MRJP reacted with MGO in PBS buffer, pH 7.4 after 216 h. Lane 13: MRJP reacted
with MGO in 0.2 M sodium acetate buffer, pH 4.0 after 216 h. Lane 14: No sample
loaded here but there was a sample overflow from lane 13. Lane 15: No sample
(empty)
Figure 6A: Inhibition of Cathepsin B with 0.5% MGO at pH 3.8,
incubation for 1 day. Figure 6B: Inhibition of Cathepsin B with 0.15% MGO and 0.5%
MGO modification of MRJP at 22°C (set temperature) or RT (ambient room
temperature).
[0026] Figure 7: Schematic chart of MRJP modification by acetic anhydride
(reaction with epsilon amino group), N-ethyl maleimide (NEM) (blocking of cysteine
residues) and MGO (reaction with Arg, Lys and Cys residues).
Figure 8: Cathepsin B inhibition after treatment of MRJPs with NEM,
acetic anhydride, and MGO.
[0028] Figure 9: Native PAGE Novex® 4-16% Bris-Tri gel of MGO-
modified BSA and Cathepsin B hydrolysis of MGO-modified MRJP. Cathepsin B
hydrolysis was performed on MGO-modified MRJP prepared at either pH 4.0 or 7.0.
Lane 1: Protein standard contains protein bands 20-12,000 kDa. Lane 2: BSA control
in PBS, pH 7.4, 37°C, overnight. Lane 3: MGO-modified BSA reaction in PBS pH 7.4,
37°C, overnight. Lane 4: MGO-modified BSA reaction in 0.2 M sodium acetate buffer
pH 4.0, 37°C, overnight. Lane 5: MGO-modified BSA reaction in PBS pH 7.4, room
temperature, overnight. Lane 6: MGO-modified BSA reaction in 0.2 M sodium acetate
buffer pH 4.0, room temperature, overnight. Lane 7: Desalted fraction of MGO-
modified MRJP at pH 4/9 days without Cathepsin B digestion. Lane 8: Desalted
fraction of MGO-modified MRJP at pH 4/9 days with Cathepsin B digestion. Lane 9:
Desalted fraction of MGO-modified MRJP at pH 7.5/9 days without Cathepsin B
digestion. Lane 10: Desalted fraction of MGO-modified MRJP at pH 7.5/9 days with
Cathepsin B digestion. Lanes 11 to Lane 15: Same as Lane 2 to Lane 6 but desalted
before loading into the gel.
[0029] Figure 10: Amino acid sequence of MRJP1, SEQ ID NO: 1.
Figures 11A-C: Inhibitory activity of peptides derived from the C-
terminus of MRJP1. Figure 11A: C-terminal sequence of MRJP1. MGO-modified
lysine residue is indicated by underlining. Figure 11B: Synthetic peptides tested for
Cathepsin B inhibition. Figure 11C: Cathepsin B inhibition by unmodified and MGO-
modified peptides.
Figures 12A-C: Inhibitory activity of peptides derived from the C-
terminus of MRJP2. Figure 12A: C-terminal sequence of MRJP2. MGO-modified
lysine residue is indicated by underlining. Figure 12B: Synthetic peptides tested for
Cathepsin B inhibition. Figure 12C: Cathepsin B inhibition by unmodified and MGO-
modified peptides.
Figures 13A-C: Inhibitory activity of peptides derived from the C-
terminus of MRJP3. Figure 13A: C-terminal sequence of MRJP3. MGO-modified
lysine residue is indicated by underlining. Figure 13B: Synthetic peptides tested for
Cathepsin B inhibition. Figure 13C: Cathepsin B inhibition by unmodified and MGO-
modified peptides.
Figures 14A-C: Inhibitory activity of peptides derived from the C-
terminus of MRJP4. Figure 14A: C-terminal sequence of MRJP4. MGO-modified
lysine residue is indicated by underlining. Figure 14B: Synthetic peptides tested for
Cathepsin B inhibition. Figure 14C: Cathepsin B inhibition by unmodified and MGO-
modified peptides.
Figures 15A-C: Inhibitory activity of peptides derived from the C-
terminus of MRJP5. Figure 15A: C-terminal sequence of MRJP5. MGO-modified
lysine residue is indicated by underlining. Figure 15B: Synthetic peptides tested for
Cathepsin B inhibition. Figure 15C: Cathepsin B inhibition by unmodified and MGO-
modified peptides.
Figures 16A-B: Additional peptides tested for Cathepsin B inhibition.
Figure 16A: Synthetic peptide sequences. Figure 16B: Cathepsin B inhibition by
unmodified and MGO-modified peptides.
DETAILED DESCRIPTION OF THE INVENTION
The following description sets forth numerous exemplary
configurations, parameters, and the like. It should be recognized, however, that such
description is not intended as a limitation on the scope of the present invention, but is
instead provided as a description of exemplary embodiments.
Definitions
In each instance herein, in descriptions, embodiments, and examples of
the present invention, the terms “comprising”, “including”, etc., are to be read
expansively, without limitation. Thus, unless the context clearly requires otherwise,
throughout the description and the claims, the words “comprise”, “comprising”, and the
like are to be construed in an inclusive sense as to opposed to an exclusive sense, that is
to say in the sense of “including but not limited to”.
“Royal jelly” is a honey bee secretion that is secreted from the glands in
the hypopharynx of worker bees. Aside from water, protein is the major component of
royal jelly and comprises the Major Royal Jelly proteins.
An “apalbumin protein” is a glycoprotein found in honey and in royal
jelly. The major apalbumin found in honey is Apalbumin 1 (Apa1) also known as Major
Royal Jelly Protein 1 (MRJP1) or royalactin. While the specification focuses on the
major apalbumins found in honey, it is to be appreciated that the other apalbumins found
in honey may also exhibit similar modification potential and similar anti-inflammatory
capacity because they are all glycoproteins with a high mannose type of glycosylation as
reported in 2000 by Kimura et al. in Biosci. Biotechnol. Biochem. There are
approximately nine major royal jelly proteins and the sequences of major royal jelly
proteins 1-9 are shown in the Sequence Listing.
[0040] It should be understood that the terms “MRJP” (e.g., any one of
MRJP1-9), “peptide” (e.g., peptide derived from any one of MRJP1-9), and “SEQ ID
NO:” (e.g., any one of SEQ ID NO: 1-145, and other such terms, for simplicity, are
used to identify the molecules described herein and not to provide their complete
characterization. Thus, a protein or peptide may be characterized herein as having a
particular amino acid sequence, a particular 2-dimensional representation of the
structure, but it is understood that the actual molecule claimed has other features,
including 3-dimensional structure, mobility about certain bonds and other properties of
the molecule as a whole. It is the molecules themselves and their properties as a whole
that are encompassed by this disclosure.
“Modification” of a primary amino acid sequence is understood to
include “deletions” (that is, polypeptides in which one or more amino acid residues are
absent), “additions” (that is, a polypeptide which has one or more additional amino acid
residues as compared to the specified polypeptide), “substitutions” (that is, a
polypeptide which results from the replacement of one or more amino acid residues),
and “fragments” (that is, a primary amino acid sequence which is identical to a portion
of the primary sequence of the specified polypeptide).
“Modified apalbumin” is to be understood to include any apalbumin or
major royal jelly protein or fragment thereof that has been modified by the chemical
reaction of methylglyoxal on the amino acids or the chemical reaction of methylglyoxal
on the side chains of the amino group that make up the protein. Methylglyoxal
modifications occur at free amino groups of lysine, arginine and/or cysteine amino acid
moieties within the apalbumin, including the terminal amino acid, and such MGO
modifications may occur on approximately 1-40 sites within the protein. For example,
modified apalbumin1 means Apa1 modified at one or more sites on its amino acid
sequences to provide a MGO-modified Apa1.
[0043] As described herein, MRJP “fragments” (i.e., fragments derived from
one or more MRJP) will be taken to include peptides obtained from any source, e.g.,
isolated naturally occurring peptides, recombinant peptides, and synthetic peptides.
These include peptides having the naturally occurring sequences as well as modified
peptide sequences. Of particular interest are functional fragments of MRJPs, i.e.,
fragments that retain one or more of the activities of the starting protein, or analogues
thereof. Such activities are described in detail herein. Thus, it will be understood that a
“fragment” is not limited to a peptide obtained directly from a polypeptide, for
example, by digestion of the original polypeptide by a peptidase.
As used herein, the term “analogue” of a protein or peptide means a
protein or peptide that includes a modification as described herein, or a peptide or
fragment thereof that includes one or more non-amino acid substituents replacing
amino acids, while the analogue still provides the necessary activity and respective
stability of the peptide or peptide fragment. The analogues of the invention may
include an acetylated N-terminus (Ac) and/or an amidated C-terminus (NH ), as well as
a C-terminal hydroxyl residue (OH). Other analogues are also possible, including those
that stabilize the domain necessary for Cathepsin B inhibition. Functional analogue are
specifically encompassed by the present invention, i.e., analogues that retain one or
more of the activities of the starting sequence. It will be understood that where a
peptide analogue is specifically noted (e.g., Ac-Xaa-Xaa-Xaa-OH; 5), the amino acid
sequence itself is also considered to be disclosed (e.g., Xaa-Xaa-Xaa). Similarly, where
an unmodified peptide is specifically noted (e.g., Xaa-Xaa-Xaa), the analogue is also
considered to be disclosed (e.g., Ac-Xaa-Xaa-Xaa-OH).
“C-terminus” or “C-terminal region” is to be understood to be the
amino acid region that is proximate the end of an amino acid chain carrying the free
alpha carboxyl group of the last amino acid.
“Lysine modified by MGO” is to be understood as a lysine amino acid
covalently bound to MGO. “Arginine modified by MGO” is understood as an arginine
amino acid covalently bound to MGO.
Amino acid “sequence similarity” or “sequence identity” refers to the
amino acid to amino acid comparison of two or more polypeptides at the appropriate
place, where amino acids are identical or possess similar chemical and/or physical
properties such as charge or hydrophobicity. Sequence similarity and identity are
typically determined by sequence alignments at the regions of highest homology.
Sequence alignment algorithms are well known and widely used in the art. Based on
the sequence comparison, a “percent identity” can be determined between the compared
polypeptide sequences.
[0048] Tissue that is “inflamed” is defined as tissue in which an immune
response has occurred in response to injury or infection in the tissue, and in which the
tissue has one or more symptoms of pain, swelling, heat, sensitivity, and redness.
As used herein, “anti-inflammatory capacity” is defined as the capacity
to clinically reduce inflammation or the symptoms of inflammation in cellular tissue.
Anti-inflammatory capacity may be determined using the phagocytosis inhibition assay
(PIA) described in PCT/NZ/2011/000271 or the DCFDA assay also described
in PCT/NZ/2011/000271 or the anti-inflammatory capacity may also be determined by
the inhibition of Cathepsin B as described in detail below.
Brief Description of the sequences
SEQ ID NO: 1 - amino acid sequence of Apa1 (also known as Major
Royal Jelly Protein 1) obtained from http://www.uniprot.org/uniprot/O18330. See
Figure 10.
SEQ ID NO: 2 - amino acid sequence of Major Royal Jelly Protein 2
obtained from http://www.uniprot.org/uniprot/O77061 is shown in the Sequence
Listing.
[0052] SEQ ID NO: 3 - amino acid sequence of Major Royal Jelly Protein 3
obtained from http://www.uniprot.org/uniprot/Q17060-1 is shown in the Sequence
Listing.
SEQ ID NO: 4 - amino acid sequence of Major Royal Jelly Protein 4
obtained from http://www.uniprot.org/uniprot/Q17060-1 is shown in the Sequence
Listing.
SEQ ID NO: 5 - amino acid sequence of Major Royal Jelly Protein 5
obtained from http://www.uniprot.org/uniprot/097432 is shown in the Sequence
Listing.
SEQ ID NO: 6 - amino acid sequence of Major Royal Jelly Protein 6
obtained from http://www.uniprot.org/uniprot/ Q6W3E3 is shown in the Sequence
Listing.
SEQ ID NO: 7 - amino acid sequence of Major Royal Jelly Protein 7
obtained from http://www.uniprot.org/uniprot/ Q6IMJ9 is shown in the Sequence
Listing.
[0057] SEQ ID NO: 8 - amino acid sequence of Major Royal Jelly Protein 8
obtained from http://www.uniprot.org/uniprot/ Q6TGR0 is shown in the Sequence
Listing.
SEQ ID NO: 9 - amino acid sequence of Major Royal Jelly Protein 9
obtained from http://www.uniprot.org/uniprot/Q4ZJX1 is shown in the Sequence
Listing.
Exemplary fragments of MRJPs include a fragment of SEQ ID NO: 1
that comprises -Lys-Ile-Ser-Ile-His-Leu (SEQ ID NO: 10) or an analogue thereof; a
fragment of SEQ ID NO: 2 that comprises -Lys-Asn-Asn-Asn-Gln-Asn-Asp-Asn (SEQ
ID NO: 11) or an analogue thereof; a fragment of SEQ ID NO: 3 that includes -Lys-
Leu-His (SEQ ID NO: 12) or an analogue thereof; a fragment of SEQ ID NO: 4: that
includes -Lys-Ser-Asn-Asn-Arg-His-Asn-Asn-Asn-Asp (SEQ ID NO: 13) or an
analogue thereof; a fragment of SEQ ID NO: 5 that includes -Lys-His-Asn-Asn (SEQ
ID NO: 14) or an analogue thereof; a fragment of SEQ ID NO: 6 that includes -Lys-
Asn-Gln-Ala-His-Leu-Asp- (SEQ ID NO: 15) or an analogue thereof; a fragment of
SEQ ID NO: 8 that includes -Lys-Asn-Thr-Arg-Cys-Ile-Ser-Pro (SEQ ID NO: 16) or
an analogue thereof; a fragment of SEQ ID NO: 9 that includes -Lys-Thr-Asn-Phe-Phe-
Ser-Ile-Phe-Leu (SEQ ID NO: 17) or an analogue thereof. In certain aspects, the
lysine residue of these fragments is modified by MGO.
Other fragments are described in detail herein, including those shown as
SEQ ID NO: 18-145. The Sequence Listing and all the sequences included therein are
hereby incorporated herein in their entirety.
MGO-modified major royal jelly proteins
Methylglyoxal or MGO is a highly chemically reactive compound with
the formula C H O . MGO is formed by multiple metabolic pathways in living
3 4 2
organisms. Certain preparations of manuka honey, which are referred to as “active”
manuka honey, contain much higher concentrations of MGO than other varieties of
honey. Active manuka honey has been determined to contain MGO concentrations up
to 1000-fold greater than the MGO concentration in other varieties of honey (E. Mavric
et al, 2008).
MGO can participate in a variety of chemical reactions in living
organisms, including the formation process of advanced glycation endproducts (AGEs).
MGO can modify proteins by reacting with the free amino groups of the amino acids
arginine, lysine, and /or cysteine and the terminal amino group, and thereby can
chemically modify proteins that contain arginine and/or lysine.
MGO-modified major royal jelly proteins (MRJPs) and fragments
thereof can be derived by isolation of the molecules from active manuka honey. The
modified proteins and fragments can be isolated from honey and/or enriched from
honey by biochemical techniques. These techniques include but are not limited to
filtration, centrifugation, and chromatography, such as ion-exchange, affinity,
hydrophobic interaction, size exclusion, and reverse-phase chromatography. MGO-
modified MRJP and fragments can also be purified from various sources or chemically
synthesized by addition of MGO to royal jelly. In another approach, the major royal
jelly may be added to honey derived from the Leptospermum genus, such as manuka
honey and jelly bush honey to achieve enrichment of MGO-modified MRJP and
fragments in the honey.
A MGO-modified MRJP or a fragment thereof may also be derived by
obtaining a sequence coding for the amino acid sequences of SEQ IDs NO: 1-145,
cloning the coding sequence into an appropriate vector, transforming a cell line with the
vector, causing the polypeptide or peptide to be expressed, purifying the polypeptide or
peptide, mixing the polypeptide or peptide with MGO to allow for chemical reaction
between MGO and the polypeptide or peptide, and purifying the MGO-modified
polypeptide or peptide.
Expression systems may contain control sequences, such as promoters,
enhancers, and termination controls such as are known in the art for a variety of hosts
(See e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold
Spring Harbor Press (1989) which is incorporated herein in its entirety). The
expression systems may also contain signal peptide and prep-pro-protein sequences that
facilitate expression of the coding sequence and/or folding of the protein.
Synthetic production or peptides may be carried out using the solid-
phase synthetic method described by Goodman M. (ed.), “Synthesis of Peptides and
Peptidomimetics” in Methods of organic chemistry (Houben-Weyl) (Workbench
Edition, 2004; Georg Thieme Verlag, Stuttgart, New York). This technique is well
understood and is a common method for preparation of peptides. Peptides may also be
synthesized using standard solution peptide synthesis methodologies, involving either
stepwise or block coupling of amino acids or peptide fragments using chemical or
enzymatic methods of amide bond formation. These solution synthesis methods are
well known in the art. See, e.g. H. D. Jakubke in The Peptides, Analysis, Synthesis,
Biology, Academic Press, New York, 1987, p. 103-165; J. D. Glass, ibid., pp. 167-184;
and EP 0324659 A2, describing enzymatic peptide synthesis methods. Commercial
peptide synthesizers, such as the Applied Biosystems Model 430A, may also be used.
MGO-modified forms of amino acid variants of MRJPs and their
fragments may also exhibit anti-inflammatory capacity. As would be understood by
one of ordinary skill in the art, minor modification of the primary amino acid sequence
of SEQ ID NO: 1 may result in a polypeptide which has substantially equivalent or
enhanced anti-inflammatory activity as compared to SEQ ID NO: 1. A peptide may be
also modified to provide substantially equivalent or enhanced anti-inflammatory
activity as the original peptide. When modification includes one or more substitutions,
preferred substitutions are those that are of a conservative nature, i.e., wherein the
residue is replaced by another of the same general type.
In making modifications to the protein or peptide, the hydropathic index
of amino acids may be considered (See, e.g., Kyte. et al., J. Mol. Biol. 157, 105-132
(1982), herein incorporated by reference in its entirety). It is known in the art that
certain amino acids may be substituted by other amino acids having a similar
hydropathic index or score and still result in a molecule having similar biological
activity.
In particular aspects, the MGO-modified MRJP variant or fragment
variant exhibits at least about 75% sequence identity to the non-variant sequence,
preferably at least about 80% identity, more preferably at least about 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any wild
type or reference sequence described herein. It is of particular interest where the MGO-
modified MRJP variant or fragment variant exhibits anti-inflammatory capacity
substantially comparable to or increased over that of non-variant molecule.
In certain aspects, the MRJP fragment is derived from the C-terminal
sequence of the protein. The fragment may be derived from the last 20 amino acids,
last 18 amino acids, last 16 amino acids, last 14 amino acids, last 12 amino acids, last
amino acids, last 8 amino acids, last 6 amino acids, last 4 amino acids, or last 2
amino acids of the C-terminal sequence of the protein. The functional fragment may
comprise 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino
acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids,
2 to 4 amino acids, or 2 amino acids of this C-terminal region. Thus, the MRJP
fragment may be 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12
amino acids, 10 amino acids, 8 amino acids, 6 amino acids, 4 amino acids, or 2 amino
acids in length. Longer fragments may also be useful. In particular aspects, the MGO-
modified lysine is at least three residues from the C-terminus, or no more than six
residues from the C-terminus of the protein.
[0071] Of particular interest are functional fragments of the C-terminus of
MRJPs as described in detail herein. However, other fragment of MRJPs may also be
obtained, for example, fragments corresponding to internal amino acid sequences of the
protein. In some aspects, a fragment may include a MRJP-derived amino acid sequence
which is fused to (i.e., contiguous with) one or more heterologous amino acid
sequences. Thus, an MRJP fragment may be presented as a portion of a major royal
jelly protein that is included as part of a larger peptide or polypeptide. This may
provide better stability and or expression capabilities, or provide options for cleavage or
tagging. In certain aspects, the MRJP fragment may be chemically linked to other
molecules, for example, one or more chemical moieties. While MGO-modified
fragments are specifically embraced, other chemical modifications of are also possible.
The MRJP fragment may also be linked to a substrate such as beads, catheters, needles,
sutures, stents, implantable medical devices, contact lenses, root canal fillers, wound
dressings, burn dressings, tissue culture plates, fibers, and paper. The fragment may be
prepared as a peptide conjugate in accordance with known methods.
[0072] The formation of MGO-modified MRJP or its fragments in honey can
be stimulated by (i) prolonged storage at ambient temperature, or (ii) incubation of
honey at elevated temperatures (30-40 Celsius), thereby increasing the anti-
inflammatory capacity of a sample of honey. Addition of MGO or an MGO precursor,
such as dihydroxyacetone (DHA) to a sample of honey, along with sufficient time
and/or heating to convert the MGO precursor to MGO, may also stimulate the
formation of MGO-modified MRJP or fragments thereof in that sample of honey, and
may also increase the anti-inflammatory capacity of the sample of honey, by the
generation of MGO-modified MRJP in the honey sample.
MRJP1 and MRJP3 with enhanced anti-inflammatory properties can
also be formed outside of honey. Completely or partially purified MRJP has been
found to be treatable with MGO, in order to yield MGO-modified MRJP. The MGO-
modified MRJP1 and MRJP3 exhibits enhanced anti-inflammatory properties when
compared to the unmodified MRJP1 and MRJP3.
MGO-modified MRJP, fragments thereof, and variants thereof may be
included in therapeutically-effective amounts in pharmaceutical compositions. It is also
to be appreciated that a peptide having MGO-modified lysine and/or arginine may be
prepared synthetically for use in the compositions.
The pharmaceutical compositions of the present disclosure may be
specially formulated for administration in solid or liquid form, including those adapted
for the following: oral administration, for example, drenches (aqueous or non-aqueous
solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and
systemic absorption, boluses, powders, granules, pastes for application to the tongue;
parenteral administration, for example, by subcutaneous, intravitreal, intramuscular,
intravenous or epidural injection as, for example, a sterile solution or suspension, or
sustained release formulation.
Also encompassed is topical application, for example, as an eye drop, or
as a cream, ointment, or a controlled release patch or spray applied to the skin; as well
as administration intravaginally or intrarectally, for example, as a pessary, cream or
foam; administration sublingually; administration ocularly; administration
transdermally; administration pulmonarily, or nasally.
When the compounds of the present invention are administered as
pharmaceuticals, to humans and animals, they can be given per se or as a
pharmaceutical composition containing, for example, about 0.1 to 99%, or about 1 to
50%, or about 10 to 40%, or about 10 to 30, or about 10 to 20%, or about 10 to 15% of
active ingredient in combination with a pharmaceutically acceptable carrier or
excipient.
[0078] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can
also be present in the pharmaceutical compositions described herein. These
compositions may also contain adjuvants such as preservatives, wetting agents,
emulsifying agents, and dispersing agents.
[0079] Prevention of the action of microorganisms upon the compounds of the
present invention may be ensured by the inclusion of various antibacterial and
antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the inclusion of agents that
delay absorption such as aluminum monostearate and gelatin.
Methods of use for MGO-modified MRJPs or fragments thereof
Cysteine proteases such as Cathepsin B are involved in the normal
lysosomal degradation and processing of proteins. The increased expression or
enhanced activation of cysteine proteases is associated with a number of medical
conditions. The MGO-modified MRJPs or fragments, or a MGO-modified MRJP- or
fragment-containing composition may thereby be used in the prevention, delay,
mitigation, or treatment of the following disorders.
Included are inflammatory disorders such as rheumatoid arthritis,
polyarthritis, and other inflammatory arthritides, inflammatory bowel disease, and
inflammatory bowel syndrome, inflammatory peritonitis, uveitis, sepsis, systemic
inflammatory response syndrome, multiple organ failure; cardiovascular disorders such
as ischemia reperfusion injury from transplantation and/or vascular surgery,
angiogenesis, neovascularization, acute cardiac allograft dysfunction, ischemic cardiac
damage, chemotherapy-induced myocardial suppression; musculoskeletal disorders
such as osteoarthritis, osteoporosis, muscular dystrophy, myositis; neurological
disorders such as multiple sclerosis, stroke, Alzheimer's disease, progressive multifocal
leukoencephalopathy (PML), prion-associated disorders, ataxia telangiectasia, central
nervous system injury; pulmonary disorders such as asthma, bronchitis, chronic
obstructive pulmonary disease (COPD), adult respiratory distress syndrome, Wegeneres
granulomatosis, and emphysema.
Included as well are proliferative disorders, including those involving
solid tumors, lymphomas, leukemias and other malignancies, for example, acute and
chronic myelogenous leukemia, neuronal cancer, cancer invasion and metastasis, tumor
angiogenesis, B and T cell lymphomas, acute and chronic lymphocytic leukemia,
resistance to chemotherapy, cancer associated coagulopathies (including deep venous
thrombosis, coronary artery disorder, pulmonary embolism, disseminated intravascular
coagulation), Hodgkin’s disease, carcinomas of the colon, liver, lung, breast, kidney,
stomach, pancreas, esophagus, oral pharynx, intestine, thyroid, prostate, bladder, brain;
osteosarcoma, chondrosarcoma and liposarcoma; neuroblastoma; melanoma; and
carcinomas derived from amnion and/or chorion).
Additionally included are infectious diseases and associated syndromes
such as septic shock (including Gram-negative sepsis), HIV infection and AIDS, genital
herpes, zoster, chickenpox, molluscum contagiosum, EBV infections, encephalitis
including EBV associated encephalitis, chorioretinitis, cytomegalovirus (CMV)
associated chorioretinitis or encephalitis, cytomegalovirus infections in neonates
(including related pneumonitis), opportunistic infections in immunocompromised
individuals (including AIDS and transplant patients), dysentery, hepatitis C, hepatitis
A, keratoconjunctivitis, bronchopneumonia (including pneumonia in
immunocompromised individuals), gastroenteritis, malaria, rhinovirus, polio,
enterovirus infections, common cold, aseptic meningitis, foot and mouth disease,
Klebsiella pneumoniae infection, Escherichia coli or Staphylococcus epidermidis,
leprosy bacteremia, otitis media, lambliasis, non-atopic sinusitis, and fulminant
hepatitis.
Further included are allergic, immunological, and autoimmune
disorders such as house dust mite allergy, transplant rejection, graft versus host disease,
Type 1 diabetes mellitus, autoimmune thyroiditis, psoriasis, dermatitis (e.g., contact
dermatitis), antibody-mediated autoimmune diseases, lupus erythematosus, Sjogren's
syndrome, autoimmune encephalomyelitis; kidney disorders such as polycystic kidney
disease, glomerulonephritis; as well as other disorders such as periodontal disease,
alcohol hepatitis, prostate hypertrophy, trauma, cutaneous mastocytosis, radiation- and
HIV-induced diarrhea, cachexia (including accompanying cancer and malnutrition),
and inflammation associated with wounds, e.g., puncture wounds, diabetic ulcers,
fungating wounds, cuts, bites, and surgical wounds.
Indications of note include: rheumatoid arthritis, polyarthritis,
Alzheimer’s disease, multiple sclerosis, especially relapsing-remitting multiple
sclerosis, progressive multifocal leukoencephalopathy (PML), asthma, bronchitis, adult
respiratory distress syndrome, Wegeneres granulomatosis, emphysema, and COPD,
melanoma, genital herpes, EBV infection, encephalitis, including EBV associated
encephalitis, choreoretinitis, including CMV choreoretinitis, bronchopneumonia,
gastroenteritis, uveitis, psoriasis, dermatitis, and various wounds.
In a specific aspect, the MGO-modified MRJPs or fragments thereof
may be formulated as a wound dressing. This includes any covering that can be applied
to a lesion. This encompasses infected and non-infected abrasions, cuts, bites, bums,
wounds, ulcers, abscesses, surgical wounds, fungating tumors, and pressure sores. The
lesion is preferably external, for example resulting from damage or injury to the skin.
The MGO-modified MRJP or fragment, or the MGO-modified MRJP-
or fragment-containing composition may be posited on a surface of a substrate, such as
a wound dressing substrate. The composition may include water and optionally another
solvent. For such compositions, the water and optional solvent may be allowed to
evaporate. The MGO-modified MRJP or fragment, or the MGO-modified MRJP- or
fragment-containing composition may be incorporated into a natural or synthetic fiber.
For example, the polypeptide or peptide may be incorporated on or into fibers used for
cloth, synthetic or paper dressings. Paper may be used as part of a temporary wound
dressing.
The treated substrate may include natural or synthetic materials, and
implantable devices. The biocompatibility of the substrate may be evaluated by any
suitable methodology known in the art, including one or more viability/cytotoxicity
assays known to those of ordinary skill in the art. The treated substrate may be in
contact with an aqueous environment, such as water or the inside of a patient.
Alternatively, the treated substrate may be contact with air or air and/or air borne
bacteria in an external environment or an enclosed bodily organ, such as a lung.
Thus, inflammation in tissue may be reduced by administering one or
more purified MGO-modified MRJPs or fragments, or a MGO-modified MRJP- or
fragment-containing composition to inflamed tissue. Inflammation from various
injuries, infections, and medical diseases may be treated. Included are acute and
chronic inflammatory diseases as noted above.
Regarding rheumatoid arthritis, it is noted that peptidyl fluoromethyl
ketones with the amino acid sequence Phe-Ala held constant but with variable N-
terminal groups were effective in inhibiting Cathepsin B activity in vitro and also
inhibiting the severity of inflammation and the extent of cartilage and bone damage in
adjuvant-induced arthritis. See Ahmed et al., 1992. It is considered that the MGO-
modified MRJP peptides or fragments or a MGO-modified MRJP-containing
composition of the invention can be used in similar treatments for rheumatoid arthritis.
[0091] MGO-modified MRJPs or fragments, or a MGO-modified MRJP- or
fragment-containing composition may reduce inflammation in tissue by reducing the
rate of phagocytosis by immune cells, and by blocking the mannose receptor or other
receptors on immune cells, which trigger phagocytosis. Immune cells include
macrophages, monocytes, dendritic cells, and granulocytes.
[0092] Phagocytosis is a cellular response or process of engulfing solid
particles and cellular uptake in the immune system. It is a major mechanism used to
remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral
particles are all examples of objects that may be phagocytosed or engulfed by a cell.
Phagocytosis occurs at the beginning of the inflammatory response of leukocytes to a
trigger of inflammation. Reactive oxygen species and cytokines are produced by cells
after phagocytosis to recruit and activate more phagocytes as part of a cascade of
cellular events which start with phagocytosis. Thus, inhibition of phagocytosis stops
the inflammatory response at the start of the cascade.
MGO-modified MRJPs or fragments, or an MGO-modified MRJP- or
fragment-containing composition may be administered to inflamed tissue in various
different forms. The MGO-modified molecule may be purified from other components.
For administration, it may be desirable to include one or more other types of
compounds such as pharmaceutically acceptable carriers, adjuvants, or other
therapeutic molecules. Other anti-inflammatory agents may be used for co-
administration with the MGO-modified MRJPs or fragments, or an MGO-modified
MRJP- or fragment-containing composition.
MGO-modified MRJPs or functional fragments thereof may be isolated
from active manuka honey, or from manuka honey, or any other type of honey to which
MGO or an MGO precursor has been added to modify the apalbumin, or it may be
purified from royal jelly or a system in which apalbumin is recombinantly expressed
and then treated with MGO.
MGO-modified MRJPs or fragments may be included in a composition
containing one or more other types of compounds. This includes MGO-modified
MRJPs or fragments contained in honey or a honey extract that has been enriched for
MGO-modified components, and MGO-modified MRJPs or fragments in extracts from
the recombinant production of an apalbumin or its peptides, and the chemical
modification of the apalbumin or its peptides with MGO.
MGO-modified MRJPs or fragments or an MGO-modified MRJP- or
fragment-containing composition may be administered to inflamed tissue in various
different forms, including but not limited to: creams, lotions, liquid solutions, or
poultices. MGO-modified MRJPs or fragments may also be administered to inflamed
tissue as by inclusion of MGO-modified MRJP peptides or fragments in an edible
product. Such products include but are not limited to beverages, candies, syrups,
lozenges, pills, and foods.
Methods of detecting MGO-modified MRJPs or MRJP fragments, and
characterizing properties of honey
The anti-inflammatory capacity of a sample of honey may be
determined through detection of MGO-modified MRJPs or fragments thereof. The
chemical modification of MRJPs by MGO generates molecules that exhibit Cathepsin
B inhibition activity. Because MRJPs are present in honey, by measuring the Cathepsin
B inhibition activity of a sample of honey, a measurement of the relative concentration
of MGO-modified MRJPs or fragments in the honey sample can be obtained. A
measurement of the concentration of MGO-modified MRJPs or fragments in a sample
of honey directly relates to the anti-inflammatory capacity of the sample of honey.
Because high MGO concentration is a feature unique to manuka honey
among all honey varieties, honey producers may try to simulate active manuka honey
by adding MGO to samples of honey that do not naturally contain a desired
concentration of MGO. Consumers prefer naturally occurring honeys over treated
honeys. Purified, active MGO is readily available from commercial chemical
producers (e.g. Sigma-Aldrich, St. Louis, MO, sells a solution of ~ 40% methylglyoxal
in water), and honey producers may add MGO to a sample of honey that does not
naturally contain a desired concentration of MGO, in order to raise the concentration of
MGO in the honey sample to a desired level.
In particular aspects, the MGO-modified MRJPs or functional
fragments thereof are obtained by one or more processes independent from natural
honey formation. A process independent from natural honey formation includes any
activity not performed by bees, and it therefore includes activities such as addition of
purified MGO to a honey sample. A process independent from natural honey formation
does not include activities such as bees collecting nectars, pollens, or other plant
products that contain high levels of MGO or MGO-precursor molecules.
The Cathepsin B inhibition activity of honey may also be used to
determine an appropriate time to harvest honey from a hive or to store harvested honey
in order to obtain honey with desired anti-inflammatory properties. Because the
modification of MRJPs by MGO in honey may occur over a period of time, a honey
producer may choose to keep honey in the hive until it contains a desired anti-
inflammatory capacity and concentration of MGO-modified MRJPs. By measuring the
Cathepsin B inhibition activity of samples of honey from the hive at different time
intervals, a honey producer can use the measurement of the Cathepsin B inhibition
activity of the honey as a method for determining the optimal time to harvest honey
from the hive in order to obtain a honey having desired anti-inflammatory properties in
the honey.
[00101] Similarly, a honey producer may also measure the Cathepsin B
inhibition activity of honey stored outside of the hive, in order to determine if the honey
sample has a desired level of anti-inflammatory properties. By measuring the
Cathepsin B inhibition activity of honey samples, a honey producer seeking to obtain a
honey sample containing a desired anti-inflammatory capacity can store honey until it
has developed a desired level of anti-inflammatory capacity by the formation of MGO-
modified MRJPs.
EXAMPLES
The examples described herein are provided for the purpose of illustrating specific
embodiments of the invention and are not intended to limit the invention in any way.
Persons of ordinary skill can utilize the disclosures and teachings herein to produce
other embodiments and variations without undue experimentation. All such
embodiments and variations are considered to be part of this invention.
Example 1: Preparation and analysis of MGO-modified MRJP proteins 1 and 3
Royal jelly obtained from Watson & Son/ManukaMed, was dissolved at
50 mg/mL in phosphate buffered saline pH 7.4 or in 100 mM disodium hydrogen
phosphate pH 9.4. The pH value of the final solutions was 3.5-4.0 when PBS was used,
or pH 7.4 when 100 mM disodium hydrogen phosphate pH 9.4 was used.
The protein solution was mixed for 10 minutes before centrifugation of
the solution at 10,000 rpm in SS34 rotor at 4 °C. The supernatant was passed through a
0.45 micron filter and then further fractionated using ion exchange chromatography and
heparin Sepharose™ to purify MRJP1. Alternatively the crude mixture was
fractionated using a 5 mL HiTrap™ desalting column (GE), or separated using size
exclusion chromatography on a Sephadex™ G75 column (GE).
The crude mixture was also reacted with MGO at final concentrations
of 0.05%, 0.1%, 0.15%, 0.5%, and 1% at pH 7.4 and 3.5-4.0 as well as pH 6.9, and pH
5.0. Reactions were performed at room temperature, 22°C, 37°C, 40°C and 60°C. The
reactions with respect of MGO coupling were performed over the following time
periods: 1 day, 2 day, 3 day, 4 day, 5 day, 6 days, 7 days, 8 days, 9 days and 14 days.
Analysis of the MGO-modified proteins was performed using native
PAGE analysis, SDS PAGE analysis, absorbance at 280 nm (protein concentration),
absorbance at 330 nm (Arg-MGO adduct formation), size exclusion chromatography
(SEC) on Sephadex™ G75, HiTrap™ desalting column and reactions with
trinitrobenzene sulphonic acid (TNBS) reaction with lysine residues, fluorescence
analysis excitation at 330 nm and emission at 410 nm (Arg-MGO adduct formation)
due to the formation of argpyrimidine (Kim et al ., 2010).
[00106] SDS PAGE analysis was performed on a Bolt® 4-12% Bis-Tris Plus
gel. A 10 well system (BGO4412BOX, Life technologies, NZ) was used in the gel
electrophoresis analysis of samples following the manufacturer’s protocol. 2-
mercaptoethanol (M3148, Sigma Aldrich, NZ) was used as a reducing agent instead of
DTT. The protein marker was SeeBlue Plus2 Pre-stained Standard (LC5925, Life
Technologies, NZ). Further SDS PAGE analysis was performed using NuPAGE®
Novex® 4-12% Bis-Tris gels, 1.0 mm, 10 well (NP0321BOX, Life technologies, NZ).
Gel electrophoresis analysis was carried out for samples dissolved in 6M urea followed
by manufacturer’s protocol. As before, 2-mercaptoethanol (M3148, Sigma Aldrich,
NZ) was used as a reducing agent instead of DTT, and the protein marker was SeeBlue
Plus2 Pre-stained Standard (LC5925, Life Technologies, NZ). This analysis revealed
the presence of two major royal jelly proteins MRJP1 and MRJP3 (Figures 1A and 1B).
MGO modification reactions were carried out at different pH conditions
for a total of 9 days, as detailed above. Samples were collected every 24 hours and
analyzed on 4-16% Native PAGE Novex® Bis-Tris gels 1.0 mm, 15 well
(BN1004BOX, Life Technologies, NZ) following instructions provided in the manual.
Typically, 4 µL of NuPAGE™ LDS sample buffer (4X) (Invitrogen, Carlsbad USA
Cat. No. NP0007) was mixed with 11 µl of protein sample plus 1 µl of Native PAGE
% G-250 sample additive. Then, 12 µl of this sample mix was loaded into native
PAGE gels. Gels were run at 150 V for 120 minutes following manufacturer’s
protocols. Protein bands were visualized after de-staining for 2-4 hours following
manufacturer's instructions. Gels were scanned using the UVITEC Cambridge, UK
Gel-doc system.
The native PAGE results are presented in Figures 2-5. At pH 4.0 but
not at pH 7.0, the formation of MRJP high molecular weight adducts is seen after 24
hours of MGO treatment (Figure 2, Lane 7). At day 9, it is apparent that the majority of
the 300 kDa hexamer is the likely source for the higher molecular weight adducts
(Figure 5, Lane 13).
Changes in the lower molecular weight species are less apparent, but it
is expected that MGO modification on the royalactin monomeric form of MRJP1 has
also occurred. It may not be as readily apparent due to preclusion of higher molecular
weight adducts from the gel. Visible precipitate was observed on the bottom of the
micro centrifuge tube upon the centrifugation.
Example 2: Incubation of major royal jelly proteins in the presence of MGO and
resulting inhibitory activity
After partial thawing, the MRJP mixture was dissolved in PBS (pH 7.4)
at a concentration of 50 mg/ml. This mixture was stirred for 30 to 60 minutes. The
resulting cloudy solution was clarified by centrifugation (10,000 rpm for 10 minutes).
The protein content was determined as 5.0 ± 0.5 mg/ml. The pH of the solution was
3.8.
For the modification with MGO, 900 μl protein solution was mixed
with 100 μl aqueous MGO solution (5 %) The 5% MGO stock solution was produced
by adding 125 µL of 40% MGO to 875 µL of water. To obtain the final MGO
concentration of 0.15% reaction, 30 µL of 5% MGO was added to 970 µL of protein
solution and incubated at 22 C (set temperature) or allowed to sit out at ambient room
temperature (RT) for 1 to 10 days. The set temperature of 22 C was maintained using a
water bath.
[00112] After each incubation interval, unreacted MGO was removed on a
HiTrap™ desalting column (5 ml, GE healthcare). The protein was eluted with PBS
buffer (25 mM, pH 7.4) at a flow rate of 2 ml/min. Eluting compounds were detected
by UV absorbance at 214, 280 and 330 nm. Alternatively, the elution was carried out
in sodium acetate buffer (10 mM, pH 4.0). The chromatography was carried out on an
Äkta-900™ system (GE-healthcare) under the control of UNICORN software.
To test for activity, MGO was removed by passing 500 µl of reaction
mixture (MRJP + MGO) through a 5 ml HiTrap™ desalting column. 1 ml fractions
were collected and tested in the DCFDA oxidation assay (see Carter WO, Narayanan
PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in
endothelial cells. J Leukoc Biol. 1994 Feb;55(2):253-8) and in the Cathepsin B assay
(see below) to determine for the presence of reactive species as well as Cathepsin B
inhibitors.
The desalting fractions 1-3 containing the high molecular weight
proteins were also analyzed for the presence of free amines using TNBS and
absorbance at 280 and 330 nm determined as well as fluorescence profile. The
numbers were normalized to protein concentration and difference between the starting
MRJP and MGO-modified MRJP determined to identify amino acids involved in the
activity associated with Cathepsin B inhibition.
Activity is observed after overnight treatment under the following
conditions: 0.5% MGO, room temperature incubation at pH 3.8, with 5 mg/ml MRJP
(Figure 6A). Such treatment produces the most rapid production of Cathepsin B
inhibitory activity. Inhibition of Cathepsin B is also found using modification with
0.15% MGO and 0.5% MGO at room temperature. See Figure 6B. When the lower
MGO concentration of 0.15% is used for MRJP modification, activity is only apparent
after longer incubation periods. The reaction products were separated using GE
Healthcare desalting column and the high MW fractions that were free of MGO were
tested for cathepsin B activity. The gel filtration was performed either at pH 7.5 in PBS
or at pH 4.5 in 100 mM sodium acetate buffer. Fractionation was performed under
conditions that corresponded to the conditions used for MGO modification either at pH
7.5 or at pH 3.5. This is in contrast to the higher MGO concentration. A similar trend
was observed for pH, whereby acidic pH produced inhibitory activity at a faster rate by
comparison to pH 7.4. See Figure 6B.
Example 3: Assay for Cathepsin B activity
Cathepsin B was obtained from Sigma (C6286-25UN Cathepsin B from
bovine spleen). The enzyme was prepared by adding 1 mL of 100 mM sodium acetate
pH 4.5 containing 2 mM EDTA and 2 mM 2-mercaptoethanol to the enzyme vial. This
activated the Cathepsin B. For assays, 50 µl of activated Cathepsin B was diluted with
mL of the assay buffer (100 mM sodium acetate pH 4.5 containing 2 mM EDTA and
2 mM 2-mercaptoethanol). 50 µl was used per well in a 96 well plate.
[00117] Assays were performed in duplicate and repeated in replicates of four
for active material. To each well of a 96 well plate, 100 µl of assay buffer was added,
followed by 50 µl of sample and 50 µl of Cathepsin B (stock 5.25 mg/mL having 12.5
U/mg) diluted 1:100 to 52.5 µg/mL for use in the assay. Total amount of Cathepsin B
in the assay was 2.625 µg. The plate was allowed to incubate at 37 °C for up to 10
minutes. However, routine analysis was performed with 2-5 minutes of incubation.
The addition of the substrate was used to initiate the reaction, Na-CBZ-
L-lysine p-nitrophenyl ester (CLN) (Sigma C 3637) (Z-Lys-pNP). The protocol was
performed similar to that described by O’Neil et al., 1996. 27 mg of substrate was
dissolved in 1 mL of DMSO and then 50 µl of the stock substrate solution was mixed
with 5 mL of assay buffer (100 mM sodium acetate pH 4.5 containing 2 mM EDTA
and 2 mM 2-mercaptoethanol). 50 µl of the substrate was added to each well of a 96
well plate. A row of eight wells of the 96 well plate was assayed during each run.
The plate was read in a SpectraMax® M4 plate reader incubated at 37
°C. The plate was agitated for 10 seconds and for 3 seconds between each read. The
Vmax rate change in absorbance at 330 nm was read over 2 minutes with 10 s interval
between reads. A delay of 30 seconds was used for the analysis. The linear portion of
the curve was used to calculate the Vmax (milli units/min). The average of duplicate
readings was performed. Active fractions were tested again to confirm activity.
Example 4: Identification of amino acid residues involved in Cathepsin B
inhibition
The following methods were employed to demonstrate which amino
acids residues were important for Cathepsin B inhibition. The MRJP were either
treated with MGO (product 77), acetic anhydride (blocking lysine residues) (product
83) or N-ethylmaleimide (NEM) (product 81). These last two samples were then further
treated with MGO (yielding products 89 and 82, respectively). A sample of the
acetylated MRJP (product 83) was taken and further reacted with NEM (yielding
product 84). This sample was then further treated with MGO (yielding product 85).
See reaction schematic in Figure 7.
Modification of MRJP mixture with MGO to give product 77
After partially thawing, the MRJP mixture was dissolved in PBS (pH
7.4) at a concentration of 50 mg/ml by stirring for 30 to 60 minutes. The resulting
cloudy solution was clarified by centrifugation (10,000 rpm for 10 minutes). The
protein content was determined as 5.0 ± 0.5 mg/ml. The pH of the solution was 3.8.
For the modification with MGO, 900 μl protein solution was mixed
with 100 μl aqueous MGO solution (5 %) and incubated at 22 C for 1 to 10 days. The
temperature was maintained by incubation in a water bath.
[00123] After each incubation interval, unreacted MGO was removed on a
HiTrap™ desalting column (5 ml, GE healthcare). The protein was eluted with PBS
buffer (25 mM, pH 7.4) at a flow rate of 2 ml/min. Eluting compounds were detected
by UV absorbance at 214, 280 and 330 nm. Alternatively, the elution was carried out in
sodium acetate buffer (10 mM, pH 4.0). The chromatography was carried out on an
Äkta-900™ system (GE-healthcare) under the control of the UNICORN software.
Modification of MRJP mixture with NEM to give product 81
The MRJP mixture was dissolved in sodium phosphate buffer (pH 9.6)
at a concentration of 50 mg/ml by stirring for 30 to 60 minutes. The resulting slightly
cloudy solution was clarified by centrifugation (10,000 rpm for 10 minutes). The
protein content was determined as 5.0 ± 0.5 mg/ml. The pH of the solution was 7.5.
[00125] To 1 ml of the protein solution 53 μl of NEM solution (200 mM in
PBS) were added and the mixture was stirred for 1 hour at room temperature.
Unreacted NEM was removed on a HiTrap™ desalting column (5 ml, GE healthcare).
The protein was eluted with sodium acetate buffer (10 mM, pH 4.0) at a flow rate of 2
ml/min. Eluting compounds were detected by UV absorbance at 214, 280 and 330 nm.
Modification of NEM-treated MRJP with MGO to give product 82
Protein containing fraction from the desalting column (fraction 2) was
modified with MGO as described above. Briefly, 900 μl of fraction 2 were reacted with
100 μl MGO (5 %) at 22 C overnight. Unreacted MGO was removed on a HiTrap™
desalting column (5 ml, GE healthcare). The protein was eluted with PBS buffer (25
mM, pH 7.4) at a flow rate of 2 ml/min. Eluting compounds were detected by UV
absorbance at 214, 280 and 330 nm. Alternatively, the elution was carried out in
sodium acetate buffer (10 mM, pH 4.0).
Acetylation of MRJP mixture to give product 83
1.2814 g of defrosted RJP mixture was dissolved in 50 mL of Tris/HCl
(0.1 M, pH 8.5) containing 6 M urea. The solution was cooled down on an ice water
bath. Acetic anhydride was added every 20 minutes in aliquots of 0.5 ml over a period
of 2 hours. The pH of the solution was determined before every addition and adjusted
to a pH above 7.5 using Tris/HCl buffer (1 M, pH 8.5).
The sample was transferred to a dialysis tubing (molecular weight cut
off 10 kDa) and dialysed over night against 1.6 litres of water with three changes of
dialysate. The content of the dialysis tubing was frozen and freeze dried. 239.9 mg of a
white slightly sticky material was recovered.
Modification of acetylated MRJP mixture with NEM to give product 84
The concentrated protein solution (500 μl) was diluted with 500 μl PBS
(25 mM, pH 7.4), 53 μl of NEM solution (200 mM in PBS) were added and the mixture
was stirred for 1 hour at room temperature. Unreacted NEM was removed on a
HiTrap™ desalting column (5 ml, GE healthcare). The protein was eluted with sodium
acetate buffer (10 mM, pH 4.0) at a flow rate of 2 ml/min. Eluting compounds were
detected by UV absorbance at 214, 280 and 330 nm.
Modification of NEM-treated acetylated MRJP with MGO to give product 85
Protein containing fraction from the desalting column (fraction 2) was
modified with MGO as described above. Briefly, 900 μl of fraction 2 were reacted with
100 μl MGO (5 %) at 22 C overnight. Unreacted MGO was removed on a HiTrap™
desalting column (5 ml, GE healthcare). The protein was eluted with PBS buffer (25
mM, pH 7.4) at a flow rate of 2 ml/min. Eluting compounds were detected by UV
absorbance at 214, 280 and 330 nm. Alternatively, the elution was carried out in
sodium acetate buffer (10 mM, pH 4.0).
Results from NEM, acetylation, and MGO modifications
The samples after treatment with MGO were desalted with a 5 mL
HiTrap™ desalting column and fraction 2 containing the high molecular weight
proteins were analysed for their ability to inhibit Cathepsin B. The assay for Cathepsin
B activity is detailed further above.
MRJP treated with MGO produced inhibitory activity (Figure 8).
Inhibitory activity was also observed for the NEM plus MGO treated MRJP sample.
However, upon acetylating MRJP and treating with MGO (product 89; see schematic in
Figure 7), all inhibitory activity was lost, providing evidence that MGO modification of
lysine residues is important for Cathepsin B inhibitory activity (Figure 8). After
acetylation of the lysine residues of MRJP, the proteins appeared to stimulate rather
than inhibit Cathepsin B activity (Figure 8).
[00133] It is noted that NEM is also a cysteine protease inhibitor. Our results
showed that NEM modification followed by MGO modification produced greater
inhibition of Cathepsin B. This was attributed to residual NEM remaining in the
samples of MGO-modified protein, allowing the NEM to act in combination with the
MGO-modified protein and to further inhibit Cathepsin B activity.
Thus, in summary, MGO reaction with MRJP produces a Cathepsin B
inhibitor. Blocking thiol groups with NEM followed by MGO treatment also produces
an active inhibitor of Cathepsin B. Acetylation of MRJP followed by reaction with
MGO prevented the production of the Cathepsin B inhibitory activity. Acetylation
followed by NEM treatment to block both lysine and cysteine residues on MRJPs
followed by MGO treatment also failed to produce an active Cathepsin B inhibitor.
[00135] It has been demonstrated in PCT/NZ/2011/000271 that acetylation
selectively blocks lysine residues. As noted above, the acetylated MRJP sample was
found to lack activity after addition of MGO. This indicated that the MGO
modification of lysine residues is important for the functional activity seen by modified
MRJP in relation to the formation of a reactive form of MGO on the surface of the
protein.
To confirm these results, another sample prepared in an identical
fashion was tested for its ability to inhibit Cathepsin B. The acetylated proteins from
royal jelly were unable to inhibit Cathepsin B, even after optimal MGO treatment, i.e.,
0.5% MGO at pH 3.5, 22 °C overnight. The speed by which this could be done makes
it preferred over the other methods outlined in Figure 6B which took from 3-7 days to
generate similar levels of Cathepsin B inhibitory activity. It was concluded that MGO
modification of lysine is a key factor in Cathepsin B inhibitory activity.
Example 5: Cathepsin B digestion of MGO-modified MRJP
Cathepsin B working reagent solution was prepared (1:50) from
Cathepsin B stock solution in the buffer system containing 100 mM sodium acetate, 2
mM EDTA and 2.5 mM 2-mercaptoethanol, pH 5.0.
Cathepsin digestion was performed on selected samples of SEC
fractions. Typically, 10 µl of Cathepsin B working solution was mixed with 20 µl of
protein sample and digested at 37°C for overnight. After hydrolysis, the digests were
prepared for native PAGE analysis. The gel was stained with Coomassie® G250 as
shown in Figure 9, see Lanes 7-10.
Samples used in the Cathepsin B digestion were: 1) Crude MRJP
protein; 2) Desalted fraction (first big peak) of MGO-modified MRJP at pH 4.0 for 9
days sample; 3) Desalted fraction (first big peak) of MGO-modified MRJP in PBS (pH
7.5) for 9 days sample; 4) SEC fractions of pH 5.0 fractions; 5) SEC fractions of
reaction 221.
Results are depicted in Figure 9. Lane 1 shows native PAGE molecular
weight markers, Lane 7 shows the undigested MGO-modified MRJP at pH 4.0, Lane 8
shows the Cathepsin B digested MGO-modified MRJP at pH 4.0, Lane 9 shows
undigested MGO-modified MRJP at pH 7.0, and Lane 10 shows Cathepsin B digested
MGO-modified MRJP at pH 7.0.
At pH 4.0, which is the anticipated pH of honey, high molecular weight
adducts (>480 kDa) of MRJPs are formed (see Figure 9, Lane 7 and Figure 2, Lane 5).
These bands are not evident when the MGO modification is performed at pH 7.0 (see
Figure 9, Lanes 9-10). Thus, Cathepsin B is unable to hydrolyze the protein at pH 7.0
but was able to hydrolyze the lower molecular weight species produced at pH 4.0. The
higher molecular weight species at pH 4.0 appeared to be more resistant to proteolysis
than the lower molecular weight species. However, the species produced by MGO-
modification of the MRJP at pH 7.0 appeared also to be resistant to hydrolysis.
Without wishing to be limited by theory, the inability of Cathepsin B to
hydrolyze MRJP is proposed to be due to inhibition of this enzyme. We refer to Figure
9. In contrast, the native hexamer form of MRJP1 and the monomeric form of
royalactin were readily degraded by this enzyme (data not shown).
Notably, the level of protein loaded in the gel varied where 25% less
protein was loaded for the hydrolyzed samples. For hydrolysis, 10 µL of a 1 mg/mL
solution (10 µg) of Cathepsin B was used. Thus, a very high level of enzyme activity
was employed for these experiments. Moreover, the reaction was performed over two
days at 37°C. Under these conditions, we expected to see complete or near complete
hydrolysis of the MRJP proteins.
As Cathepsin B has an exopeptidase activity at pH 4.0, it was expected
that dipeptides would be generated from the hydrolysis that cannot be seen by native
PAGE analysis. The incomplete hydrolysis we observed suggests that some Cathepsin
B inhibition occurred, and/or the length of time and concentration of Cathepsin B was
higher than the inhibitory activity of the MGO-modified MRJP proteins. This may
have allowed partial hydrolysis of the 58 kDa protein (MRJP1) and the band present
around 242 kDa.
[00145] By comparison, the larger cross-linked complexes did not appear to be
susceptible to Cathepsin B cleavage at pH 4.0. Cross-linking of proteins with MGO
occurs between lysine residues or lysine and Arg residues. At pH 7.0, the hydrolysis of
the oligomeric complex of MRJP proteins was not hydrolyzed by Cathepsin B. This
was attributed to the substrate specificity of Cathepsin B at this pH being limited to
cleavage at Arg-Arg | Xaa sequences within the MRJP proteins, i.e., endopeptidase
activity.
Yet, this site within MRJPs does not appear to be available for
cleavage. The protein level observed on the gel after substantial hydrolysis with
Cathepsin B is essentially the same as that observed without the addition of Cathepsin
B. It is possible that Cathepsin B was completely inhibited under these conditions.
MGO modification of Arg residues is also likely. This would prevent recognition of
the MRJP cleavage site for Cathepsin B at pH 7.0. It is also possible that the native gel
electrophoresis failed to separate the protein fragments sufficiently.
Example 6: Proposed mechanism of Cathepsin B inhibition
[00147] Cathepsin B is a thiol protease that participates in intracellular
degradation and turnover of proteins. It is an important enzyme in the phagocytosis
process and it has also been implicated in tumor invasion and metastasis.
The primary specificity of Cathepsin B is for cleavage sites Arg-Arg- ǀ-
Xaa (where ǀ indicates the point of cleavage). The MRJPs would be predicted to be
suitable substrates for this enzyme due to the presence of an Arg-Arg peptide sequence
in MRJP1-5. However, the significance of this cleavage site has been questioned by
our results. In particular, MGO-modified bovine serum albumin that has the same Arg-
Arg peptide sequence shows no anti-inflammatory activity, in contrast to the MGO-
modified MRJP1 and MRJP3 proteins (results not shown). The results indicate that
Arg-Arg cleavage may only be relevant for plasma membrane bound Cathepsin B at
optimal conditions of pH 7 (data not shown).
Cathepsin B also has some C-terminal dipeptidase activity (Brömme et
al., 1987 and Chapman et al., 1994). Without wishing to be bound by theory, it is
proposed that the C-terminal peptide regions of MGO-modified MRJP1 and MGO-
modified MRJP3 are inhibiting the activity of Cathepsin B. It is proposed that the
MGO-modified lysine, which can be involved in thiol lysine cross-links, is in close
proximity to the thiol active site of Cathepsin B. In this way, the MGO on the lysine is
reacting with the thiol active site of Cathepsin B, and inhibiting its action.
Notably, MRJP1 has a lysine (see SEQ ID NO: 1 at 427) that is the
third cleavage site from the C-terminus end. MRJP3 has a lysine residue at the first
cleavage site at 542 (see SEQ ID NO: 3). Similarly, MRJP2 has a lysine residue at the
fourth cleavage site at 445 (see SEQ ID NO: 2) and MRJP 4 has a lysine residue at the
fifth cleavage site at 455 (see SEQ ID NO: 4). MRJP 5 has a lysine residue at the
second cleavage site at 595 (SEQ ID NO: 5). MRJP6 has a lysine residue at the 3rd
cleavage site at 431 (see SEQ ID NO: 6). MRJP8 has a lysine at the 4th cleavage site at
409 (see SEQ ID NO: 8) and MRJP 9 has a lysine at the fourth cleavage site at 415 (see
SEQ ID NO: 9).
Bovine serum albumin does not have a lysine at the C-terminus until the
fifth cleavage point of Cathepsin B. In this way, the lysine residue in MGO-modified
BSA may be too far away to inhibit Cathepsin B. Moreover, the lysine residue at 573
in BSA is involved in an alpha helical structure and not readily available for cleavage
by Cathepsin B. It is therefore postulated that the inhibitory activity of MGO-modified
MRJP1 and MRJP3 is seen because of the relatively close proximity of the lysine to the
C-terminus end of the peptide. It is anticipated also that MGO-modified MRJP2,
MRJP4, MRJP5, MRJP6, MRJP8, and MRJP9 will also show Cathepsin B inhibitory
activity.
The presence of a reactive MGO species on the surface of the protein
was also detected using DCFDA oxidation to fluorescein (data not shown). The
reaction of hydrogen peroxide with MGO led to a reaction in the level of DCFDA
oxidized and therefore leads to a reduction in the level of fluorescence detected (data
not shown). Isolation of high molecular weight proteins using gel filtration or
HiTrap™ desalting led to the identification of a highly reactive species on the surface
of the protein. This species as seen by DCFDA analysis correlates to the Cathepsin B
inhibitory activity (data not shown).
It is known that the Arg-MGO adduct formation is favored under lower
MGO concentrations and this leads to an irreversible adduct that can be detected with
absorbance at 330 nm or fluorescence Ex330nm and Em410nm. The reaction with Cys
residues forms a hemiacetyl that is reversible and unstable leading to release of MGO
off the surface of the protein. The reaction with lysine can form both reversible and
irreversible reactions with MGO.
The initial adduct formed is slowly rearranged to form a stabilized
covalently linked MGO attached to the epsilon amino group of lysine. Further
rearrangements can occur, leading to the generation of various chemical functionality
including N(ɛ)-(carboxyethyl)lysine (CEL) acid group, hydroxyl group, aldehyde or
ketone functionality as well as the irreversible reactions with other protein to form
cross-links.
[00155] MGO modification can lead to reactive species such as aldehydes
bound to the protein, and aldehydes can react with the thiol of the Cathepsin B active
site. From this, it is possible that inhibition of Cathepsin B may occur between MGO-
modified lysine residues with an aldehyde functionality. Lysines are also of interest in
Cathepsin B inhibition, as bound MGO can undergo a cannizzaro rearrangement
forming CEL and a hydroxyl group, which has carboxylic acid functionality. Two
aldehydes react, where one aldehyde is reduced to a corresponding alcohol, while the
second aldehyde is oxidized to carboxylic acid.
At pH 4.0, Cathepsin B has a C-terminal exodipeptidase activity. It is
hypothesized that the acid functionality present in the CEL, carboxylic acid moiety may
be recognized by Cathepsin B as a C-terminus. This would place the peptide in an
orientation that prevents cleavage. Both structures are potentially formed by MGO
upon reaction with lysine. Various reaction pathways are likely to be favored under
various conditions.
The chemical reactivity of MGO under acidic conditions, as present in
Manuka honey, appears to enable formation of high molecular weight cross-links. This
would appear to favor aldehyde formation, which, in turn, continues to react to form
protein cross-links. The cannizzaro rearrangement is favored under basic conditions,
which is more likely to occur when MRJP proteins are reacted at neutral pH 7.0. The
cannizzaro rearrangement prevents further cross-linking between proteins. The
aldehyde functional group that is involved in cross-linking is replaced with an acid
group and a hydroxyl group. The hydroxyl provides further diversification of the
chemistry on the surface of lysine residues after MGO modification and this can
provide changes in inhibitory selectivity and affinity.
As noted above, we observed that blocking lysine residues with acetic
anhydride followed by MGO-modification blocks Cathepsin B inhibition. This
highlights the importance of lysines in producing inhibitory activity. The reaction of
lysine epsilon amino groups with acetic anhydride produces an amide bond. This
removes the features required for recognition by the C-terminal exopeptidase activity of
Cathepsin B. This, in turn, abolishes enzyme interaction and inhibition.
Example 7: MGO modification of synthetic peptides and their inhibitory activity
[00159] Thirty-seven synthetic peptides were supplied by Mimotopes Pty Ltd
(Melbourne, Australia). The structures were based on peptide sequences in MRJP 1-5.
Particular focus was placed on the C-terminus of these proteins.
MGO modification was performed as follows. The incubation mixture
included 180 µl peptide solution (5 mM) and 20 µl MGO solution (5%). Incubation
was carried out overnight at room temperature. MGO was removed using C18 SPE
cartridges. Eluted peptides were concentrated and solutions made up to original
concentration.
The test for inhibition of Cathepsin B activity was carried out with
solutions containing 2 mM of unmodified or modified peptide according to the
previously described procedure. The synthetic peptides and results are shown in
Figures 11A-C, 12A-C, 13A-C, 14A-C, and 15A-C, and Tables 1-5, below.
Table 1: Synthetic peptides derived from the C-terminus of MRJP1
peptide SEQ ID NO: unmodified MGO-modified
Ac-PFKI-OH 22 152.05 ± 13.20 91.45 ± 0.11
Ac-KISI-OH 21 133.11 ± 4.29 92.82 ± 5.44
Ac-KISIHL-OH 20 149.04 ± 9.65 73.23 ± 17.90
Ac-PFKISIHL-OH 19 144.22 ± 12.20 28.11 ± 0.06
Table 2: Synthetic peptides derived from the C-terminus of MRJP2
peptide SEQ ID NO: unmodified MGO-modified
Ac-KN-OH 28 149.55 ± 4.42 111.84 ± 6.21
Ac-NQKN-OH 27 282.39 ± 19.66 102.36 ± 6.36
Ac-NQKNNN-OH 26 146.89 ± 14.87 87.48 ± 4.31
Ac-NQKNNNQN-OH 25 290.25 ± 12.34 171.38 ± 9.10
Ac-NQKNNNQNDN-OH 24 254.04 ± 22.31 123.18 ± 12.85
Table 3: Synthetic peptides derived from the C-terminus of MRJP3
peptide SEQ ID NO: unmodified MGO-modified
Ac-HSSKLH-OH 30 121.50 ± 2.05 102.36 ± 6.36
Ac-SKLH-OH 31 146.35 ± 2.91 67.57 ± 1.73
Ac-SK-OH 32 137.62 ± 9.65 115.94 ± 1.24
Ac-HSSK-OH 33 164.77 ± 3.87 134.68 ± 1.13
Ac-SSKS-OH 34 118.71 ± 9.09 107.49 ± 4.29
Table 4: Synthetic peptides derived from the C-terminus of MRJP4
peptide SEQ ID NO: unmodified MGO-modified
Ac-KS-OH 41 135.26 ± 7.09 110.69 ± 0.89
Ac-SSKSNNRHNNND-OH 36 164.01 ± 8.57 109.55 ± 3.11
Ac-SSKSNN-OH 39 129.06 ± 6.31 107.043 ± 8.64
Ac-SSKSNNRH-OH 38 125.43 ± 9.56 101.92 ± 6.31
Ac-SSKSNNRHNN-OH 37 131.11 ± 6.491 104.11 ± 7.46
Table 5: Synthetic peptides derived from the C-terminus of MRJP5
peptide SEQ ID NO: unmodified MGO-modified
Ac-KH-OH 45 230.49 ± 16.21 115.95 ± 5.94
Ac-QNKHNN-OH 43 167.90 ± 10.09 51.30 ± 5.31
Ac-KHNN-OH 44 129.26 ± 7.90 111.90 ± 2.07
Ac-QNKH-OH 46 137.67 ± 17.04 97.77 ± 3.70
From this analysis, the synthetic peptides of interest include: MRJP1:
Ac-PFKISIHL-OH (SEQ ID NO: 19); MRJP2: Ac-NQKNNNQNDN-OH (SEQ ID
NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); MRJP3: Ac-SKLH-OH (SEQ ID NO: 31);
and MRJP5: Ac-QNKHNN-OH (SEQ ID NO: 43); Ac-KH-OH (SEQ ID NO: 45).
Additional synthetic peptides were prepared based on the amino acid
sequences of MRJP1, MRJP3, and MRJP5, in accordance with the procedure noted
above.
Table 6: MRJP1 derived synthetic peptides
sample SEQ ID NO: unmodified MGO-modified
H-FDR-OH 50 174.56 ± 11.96 71.18 ± 2.22
Ac-FDR-OH 53 85.57 ± 3.51 44.78 ± 1.54
H-HNIR-OH 54 151.51± 0.62 103.38 ± 3.65
Ac-HNIR-OH 59 171.58 ± 11.29 106.82 ± 3.02
H-YINR-OH 85 142.33 ± 3.64 79.21 ± 2.12
Ac-YINR-OH 87 93.14 ± 2.42 99.05 ± 7.78
H-FTK-OH 88 147.63 ± 0.70 76.59 ± 2.24
Ac-FTK-OH 91 148.84 ± 16.13 105.35 ± 5.73
H-IFVTMLR-OH 92 80.48 ± 4.25 78.70 ± 3.67
Ac-IFVTMLR-OH 93 88.31 ± 12.62 92.63 ± 2.00
H-MQK-OH 94 127.00 ± 0.93 99.48 ± 2.95
Ac-MQK-OH 95 121.24 ± 3.57 69.15 ± 4.87
H-CDR-OH 96 165.10 ± 0.38 96.01 ± 6.45
Ac-CDR-OH 97 120.52 ± 8.17 78.51 ± 2.51
H-MTR-OH 98 143.48 ± 2.33 104.39 ± 2.68
Ac-MTR-OH 99 126.30 ± 8.12 118.90 ± 5.62
TNBS-FAK-pNA 100 99.27 ± 3.8 122.48 ± 7.65
TNBS = N-terminus blocked with trinitrobenzene sulphonic acid. pNA = paranitroanilide.
Table 7: MRJP3 derived synthetic peptides
sample SEQ ID NO: unmodified MGO-modified
H-QNDNK-OH 101 104.37 ± 4.99 68.42 ± 0.22
Ac-QNDNK-OH 102 167.26 ± 7.64 122.35 ± 3.05
H-QNDNR-OH 103 162.38 ± 16.36 116.16 ± 3.66
Ac-QNDNR-OH 104 161.75 ± 9.25 132.44 ± 4.77
H-QNGNK-OH 105 170.73 ± 5.14 118.80 ± 11.41
Ac-QNGNK-OH 106 187.34 ± 9.14 89.21 ± 10.96
H-QNGNR-OH 107 74.41 ± 4.98 84.99 ± 4.08
Ac-QNGNR-OH 108 101.30 ± 6.00 88.67 ± 4.38
Table 8: MRJP5 derived synthetic peptides
sample SEQ ID NO: unmodified MGO-modified
H-TNR-OH 109 102.61 ± 5.34 91.86 ± 3.50
Ac-TNR-OH 110 96.48 ± 2.03 91.72 ± 0.84
H-MDK-OH 111 104.25 ± 2.73 70.62 ± 12.34
Ac-MDK-OH 112 109.67 ± 3.13 99.38 ± 1.63
H-MDR-OH 113 101.73 ± 2.37 74.46 ± 0.97
Ac-MDR-OH 114 107.34 ± 1.46 88.56 ± 2.14
H-TDK-OH 115 103.18 ± 4.50 87.53 ± 3.26
Ac-TDK-OH 116 100.51 ± 10.69 92.75 ± 2.41
H-IDR-OH 117 98.02 ± 2.54 85.60 ± 5.32
Ac-IDR-OH 118 93.46 ± 12.24 81.75 ± 3.26
H-VNR-OH 119 97.75 ± 10.62 82.63 ± 7.88
Ac-VNR-OH 120 99.83 ± 3.40 68.49 ± 8.73
H-MHR-OH 121 96.77 ± 9.26 80.66 ± 6.19
Ac-MHR-OH 122 101.26 ± 4.59 90.13 ± 17.28
H-MNR-OH 123 112.08 ± 6.03 78.56 ± 5.37
Ac-MNR-OH 124 100.83 ± 2.26 86.94 ± 3.01
H-LQK-OH 125 102.47 ± 4.27 95.26 ± 5.54
Ac-LQK-OH 126 102.05 ± 10.32 90.15 ± 1.52
Further synthetic peptides derived from MRJP1-5 were tested as
described herein. The synthetic peptides and results are shown in Figures 16A and
16B, and Table 9, below.
Table 9: Additional synthetic peptides tested for inhibitory activity
peptide derived from MRJP SEQ ID NO: unmodified MGO-modified
Ac-LVK-OH MRJP3 48 143.92 ± 3.15 80.84 ± 3.07
(see Leu170)
Ac-LVK-NH MRJP3 49 92.71 ± 7.78 80.03 ± 0.10
(see Leu170)
Ac-LIR-OH MRJP2, 4 51 128.28 ± 2.50 0.07 ± 3.96
(see Leu408)
Ac-LIR-NH MRJP2, 4 52 65.39 ± 2.75 72.56 ± 9.33
(see Leu408)
Ac-LLK-OH MRJP1, 2, 4 56 141.56 ± 13.90 147.60 ± 5.98
(see Leu146;
Leu104; Leu169)
Ac-LLK-NH MRJP1, 2, 4 57 159.34 ± 2.34 136.90 ± 14.42
(see Leu146;
Leu104; Leu169)
Ac-KI-OH MRJP1-5 58 108.98 ± 9.03 92.08 ± 5.79
+ = MRJP1: Lys75, Lys358, Lys427; MRJP2: Lys121, Lys127, Lys356, Lys385;
MRJP3: Lys132, Lys361, Lys390; MRJP4: Lys131, Lys356; MRJP5: Lys100, Lys130,
Lys310, Lys518.
Another synthetic peptide is FAK (Phe-Ala-Lys; e.g., Ac-FAK-OH;
SEQ ID NO: 60) which includes a sequence found in MRJP1, MRJP3, and MRJP4.
A table to summarize the amino acid sequences from this Example is
provided as follows.
Table 10: Summary of amino acid sequences assessed for inhibitory activity
sequence SEQ ID NO: sequence SEQ ID NO: sequence SEQ ID NO:
PFKI 61 HSSKLH 70 KH 80
KISI 62 SKLH 71 QNKHNN 81
KISIHL 63 SK 72 KHNN 82
PFKISIHL 64 HSSK 73 QNKH 83
KN 65 SSKS 74 LVK 84
NQKN 66 KS 75 LIR 86
NQKNNN 67 SSKSNNRHNNND 76 LLK 89
NQKNNNQN 68 SSKSNN 77 KI 90
NQKNNNQNDN 69 SSKSNNRH 78
SSKSNNRHNN 79
sequence SEQ ID NO: sequence SEQ ID NO:
sequence SEQ ID NO:
FDR 127 MTR 134 TNR 139
HNIR 128 FAK 135 MDK 140
YINR 129 QNDNR 136 MDR 142
FTK 130 QNGNK 137 TDK 143
IFVTMLR 131 QNGNR 138 IDR 144
MQK 132 VNR 145
CDR 133 MHR 47
MNR 141
LQK 55
The results show that both C-terminal and internal peptides can provide
inhibition of Cathepsin B after modification of lysine with MGO. The peptide
analogue LIR (SEQ ID NO: 51) has particularly potent activity.
The presence of endogenous protease activity in honey has been
determined previously ((Larocca et al., 2012)), and we demonstrate that this protease
leads to degradation of MRJP proteins in the presence of urea. The release of peptides
from MRJP proteins is therefore expected during prolonged incubation of Manuka
honey leading to release of MGO-modified peptides that have enhanced Cathepsin B
inhibition.
Certain peptides show stimulatory activity in the absence of MGO
treatment. This activation is in most cases lost after treatment with MGO. In some
cases, longer peptides are more active. For specific peptides, the lysine is positioned at
least 3 amino acid away from the C-terminal amino acid, and no more than 6 amino
acids away from the C-terminal amino acid. For inhibition, a free C-terminus is
typically present. This allows a favorable interaction with Cathepsin B and potentially
increased inhibition.
For the assayed peptides, an acid functional end is more likely to
produce increased activity as compared to an amidated end. This is consistent with
internal protease digestion having the specificity similar to that of pancreatic trypsin,
i.e., cleavage C-terminal to arginine and lysine residues, as long as the following amino
acid is not proline. The preference for a C-terminal carboxylate group is
understandable in context of the structure of Cathepsin B and its exopeptidase activity
that acts on a C-terminal end.
[00172] Notably, the occlusive loop of Cathepsin B contains two histidine
residues that are believed to coordinate to the C-terminus of the peptide. See, e.g.,
Musil, D., Zucic, D., Turk, D., Engh, R.A., Mayr, I., Huber, R., Popovic, T., Turk,
V., Towatari, T., Katunuma, N., Bode, W. (1991) The refined 2.15 A X-ray crystal
structure of human liver cathepsin B: the structural basis for its specificity. EMBO J.
10: 2321-2330. This positions the peptide bond between amino acid 2 and 3 up from
the C-terminus of the peptide. In addition, the orientation of peptides with respect to
Cathepsin B may be inverted 180° if the primary amine or epsilon amino group of
lysine has reacted with MGO. As noted above, this can undergo a cannizzaro
rearrangement to produce CEL. This may also occur on the side chain epsilon amino
group of lysine.
Regarding the FAK peptide analogue (SEQ ID NO: 100), this showed
limited inhibitory activity with the C-terminus containing paranitroanilide (pNA). It
was postulated that this could block recognition of the peptide by Cathepsin B. To test
this, the pNA group was removed under basic conditions in the presence of NaOH. The
peptide was reassessed for activity before and after MGO-modification (data not
shown). Inhibitory activity was still not observed, suggesting that the position of lysine
within the peptide sequence can affect activity. From this, it is concluded that it may be
beneficial to position the lysine such that it sits closer to the active site of Cathepsin B.
The present invention and its embodiments have been described in
detail. However, the scope of the present invention is not intended to be limited to the
particular embodiments of any process, manufacture, composition of matter,
compounds, means, methods, and/or steps described in the specification. Various
modifications, substitutions, and variations can be made to the disclosed material
without departing from the scope and/or essential characteristics of the present
invention.
Accordingly, one of ordinary skill in the art will readily appreciate from
the disclosure that later modifications, substitutions, and/or variations performing
substantially the same function or achieving substantially the same result as
embodiments described herein may be utilized according to such related embodiments
of the present invention. Thus, the invention is intended to encompass, within its
scope, the modifications, substitutions, and variations to processes, manufactures,
compositions of matter, compounds, means, methods, and/or steps disclosed herein.
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Claims (53)
1. Use of a functional fragment of a major royal jelly protein (MRJP) for preparation of a medicament for reducing inflammation in a cellular tissue in a patient; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises 2 to 20 amino acids of the last 20 amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
2. The use of claim 1, wherein the functional fragment comprises 2 to 10 amino acids of the last 10 amino acids at the C-terminus of the protein.
3. The use of claim 1 or claim 2, wherein the major royal jelly protein (MRJP) is selected from the group consisting of MRJP1 (SEQ ID NO: 1), MRJP2 (SEQ ID NO: 2), MRJP3 (SEQ ID NO: 3), MRJP4 (SEQ ID NO: 4), MRJP5 (SEQ ID NO: 5), MRJP6 (SEQ ID NO: 6), MRJP7 (SEQ ID NO: 7), MRJP8 (SEQ ID NO: 8), and MRJP9 (SEQ ID NO: 9).
4. The use of any one of claims 1 to 3, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. KISIHL (SEQ ID NO: 10); ii. KNNNQNDN (SEQ ID NO: 11); iii. KLH (SEQ ID NO: 12); iv. KSNNRHNNND (SEQ ID NO: 13); v. KHNN (SEQ ID NO: 14); vi. KNQAHLD (SEQ ID NO: 15); vii. KNTRCISP (SEQ ID NO: 16); and viii. KTNFFSIFL (SEQ ID NO: 17).
5. The use of any one of claims 1 to 3, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. PFKISIHL (SEQ ID NO: 64); ii. NQKNNNQNDN (SEQ ID NO: 69); NQKN (SEQ ID NO: 66); iii. SKLH (SEQ ID NO: 71); and iv. QNKHNN (SEQ ID NO: 81); KH (SEQ ID NO: 80).
6. The use of any one of claims 1 to 3, or 5, wherein the functional fragment is a peptide analogue selected from the group consisting of: i. Ac-PFKISIHL-OH (SEQ ID NO: 19); ii. Ac-NQKNNNQNDN-OH (SEQ ID NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); iii. Ac-SKLH-OH (SEQ ID NO: 31); iv. Ac-QNKHNN-OH (SEQ ID NO: 43); and Ac-KH-OH (SEQ ID NO: 45).
7. Use of a functional fragment of a major royal jelly protein (MRJP) for preparing a medicament for reducing inflammation in a cellular tissue; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
8. The use of claim 7, wherein the functional fragment is a peptide analogue selected from the group consisting of: Ac-LVK-OH (SEQ ID NO: 48), Ac-LIR-OH (SEQ ID NO: 51), Ac-FDR-OH (SEQ ID NO: 53), Ac-HNIR-OH (SEQ ID NO: 59), H- FTK-OH (SEQ ID NO: 88), and Ac-QNGNK-OH (SEQ ID NO: 106).
9. The use of any one of claims 1 to 8, wherein the medicament reduces the rate of phagocytosis by immune system cells.
10. The use of any one of claims 1 to 8, wherein the medicament inhibits receptors for phagocytosis on immune system cells.
11. The use of any one of claims 1 to 8, wherein the medicament reduces respiratory burst and release of reactive oxygen species from inflammatory cells.
12. The use of any one of claims 1 to 11, wherein the inflammation is associated with one or more of the group consisting of: an inflammatory disorder, a cardiovascular disorder, a neurological disorder, a pulmonary disorder, a proliferative disorder, an infectious disease or associated syndrome, an allergic, immunological or autoimmune disorder, and inflammation associated with a wound.
13. The use of claim 12, wherein the inflammation is associated with one or more of the group consisting of: rheumatoid arthritis, polyarthritis, Alzheimer’s disease, progressive multifocal leukoencephalopathy (PML), multiple sclerosis, asthma, bronchitis, adult respiratory distress syndrome, Wegeneres granulomatosis, emphysema, chronic obstructive pulmonary disease (COPD), melanoma, genital herpes, Epstein–Barr virus (EBV) infection, encephalitis, EBV associated encephalitis, choreoretinitis, cytomegalovirus (CMV) associated choreoretinitis, bronchopneumonia, gastroenteritis, uveitis, psoriasis, and dermatitis.
14. The use of claim 12, wherein the wound is one or more of the group consisting of: a diabetic ulcer, fungating wound, puncture wound, cut, bite, and surgical wound.
15. Use of a functional fragment of a major royal jelly protein (MRJP) for preparing a medicament for treating rheumatoid arthritis in a subject; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises 2 to 20 amino acids of the last 20 amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
16. The use of claim 15, wherein the functional fragment comprises 2 to 10 amino acids of the last 10 amino acids at the C-terminus of the protein.
17. The use of claim 15 or claim 16, wherein the major royal jelly protein (MRJP) is selected from the group consisting of MRJP1 (SEQ ID NO: 1), MRJP2 (SEQ ID NO: 2), MRJP3 (SEQ ID NO: 3), MRJP4 (SEQ ID NO: 4), MRJP5 (SEQ ID NO: 5), MRJP6 (SEQ ID NO: 6), MRJP7 (SEQ ID NO: 7), MRJP8 (SEQ ID NO: 8), and MRJP9 (SEQ ID NO: 9).
18. The use of any one of claims 15 to 17, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. KISIHL (SEQ ID NO: 10); ii. KNNNQNDN (SEQ ID NO: 11); iii. KLH (SEQ ID NO: 12); iv. KSNNRHNNND (SEQ ID NO: 13); v. KHNN (SEQ ID NO: 14); vi. KNQAHLD (SEQ ID NO: 15); vii. KNTRCISP (SEQ ID NO: 16); and viii. KTNFFSIFL (SEQ ID NO: 17).
19. The use of any one of claims 15 to 17, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. PFKISIHL (SEQ ID NO: 64); ii. NQKNNNQNDN (SEQ ID NO: 69); NQKN (SEQ ID NO: 66); iii. SKLH (SEQ ID NO: 71); iv. QNKHNN (SEQ ID NO: 81); and KH (SEQ ID NO: 80).
20. The use of any one of claims 15 to 17, or 19, wherein the functional fragment is a peptide analogue selected from the group consisting of: i. Ac-PFKISIHL-OH (SEQ ID NO: 19); ii. Ac- NQKNNNQNDN-OH (SEQ ID NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); iii. Ac-SKLH-OH (SEQ ID NO: 31); iv. Ac-QNKHNN-OH (SEQ ID NO: 43); and Ac-KH-OH (SEQ ID NO: 45).
21. Use of a functional fragment of a major royal jelly protein (MRJP) for preparing a medicament for treating rheumatoid arthritis in a patient; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
22. The use of claim 21, wherein the functional fragment is a peptide analogue selected from the group consisting of: Ac-LVK-OH (SEQ ID NO: 48), Ac-LIR-OH (SEQ ID NO: 51), Ac-FDR-OH (SEQ ID NO: 53), Ac-HNIR-OH (SEQ ID NO: 59), H- FTK-OH (SEQ ID NO: 88), and Ac-QNGNK-OH (SEQ ID NO: 106).
23. The use of any one of claims 15 to 22, wherein administration is one or more of oral, topical, and parenteral administration.
24. The use of any one of claims 15 to 23, wherein administration is intravenous, intravitreal, or intramuscular administration.
25. An isolated functional fragment of a major royal jelly protein (MRJP), wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. KISIHL (SEQ ID NO: 10); ii. KNNNQNDN (SEQ ID NO: 11); iii. KLH (SEQ ID NO: 12); iv. KSNNRHNNND (SEQ ID NO: 13); v. KHNN (SEQ ID NO: 14); vi. KNQAHLD (SEQ ID NO: 15); vii. KNTRCISP (SEQ ID NO: 16); viii. KTNFFSIFL (SEQ ID NO: 17); and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
26. An isolated functional fragment of a major royal jelly protein (MRJP), wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. PFKISIHL (SEQ ID NO: 64); ii. NQKNNNQNDN (SEQ ID NO: 69); NQKN (SEQ ID NO: 66); iii. SKLH (SEQ ID NO: 71); iv. QNKHNN (SEQ ID NO: 81); KH (SEQ ID NO: 80); and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
27. The isolated functional fragment of claim 26, wherein the functional fragment is a peptide analogue selected from the group consisting of: i. Ac-PFKISIHL-OH (SEQ ID NO: 19); ii. Ac-NQKNNNQNDN-OH (SEQ ID NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); iii. Ac-SKLH-OH (SEQ ID NO: 31); iv. Ac-QNKHNN-OH (SEQ ID NO: 43); and Ac-KH-OH (SEQ ID NO: 45).
28. An isolated functional fragment of a major royal jelly protein (MRJP); wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
29. The isolated functional fragment of claim 28, wherein the functional fragment is a peptide analogue selected from the group consisting of: Ac-LVK-OH (SEQ ID NO: 48), Ac-LIR-OH (SEQ ID NO: 51), Ac-FDR-OH (SEQ ID NO: 53), Ac-HNIR- OH (SEQ ID NO: 59), H-FTK-OH (SEQ ID NO: 88), and Ac-QNGNK-OH (SEQ ID NO: 106).
30. The isolated functional fragment of any one of claims 25 to 29, which is isolated from Leptospermum scoparium derived manuka honey.
31. The isolated functional fragment of any one of claims 25 to 29, which is a synthetic or recombinant molecule.
32. A composition comprising the isolated functional fragment of any one of claims 25 to 31, or an analogue thereof.
33. A wound dressing comprising a functional fragment of a major royal jelly protein (MRJP); wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. KISIHL (SEQ ID NO: 10); ii. KNNNQNDN (SEQ ID NO: 11); iii. KLH (SEQ ID NO: 12); iv. KSNNRHNNND (SEQ ID NO: 13); v. KHNN (SEQ ID NO: 14); vi. KNQAHLD (SEQ ID NO: 15); vii. KNTRCISP (SEQ ID NO: 16); viii. KTNFFSIFL (SEQ ID NO: 17); and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
34. A wound dressing comprising a functional fragment of a major royal jelly protein (MRJP); wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. PFKISIHL (SEQ ID NO: 64); ii. NQKNNNQNDN (SEQ ID NO: 69); NQKN (SEQ ID NO: 66); iii. SKLH (SEQ ID NO: 71); iv. QNKHNN (SEQ ID NO: 81); KH (SEQ ID NO: 80); and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
35. The wound dressing of claim 34, wherein the functional fragment is a peptide analogue selected from the group consisting of: i. Ac-PFKISIHL-OH (SEQ ID NO: 19); ii. Ac-NQKNNNQNDN-OH (SEQ ID NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); iii. Ac-SKLH-OH (SEQ ID NO: 31); iv. Ac-QNKHNN-OH (SEQ ID NO: 43); and Ac-KH-OH (SEQ ID NO: 45);
36. A wound dressing comprising a functional fragment of a major royal jelly protein (MRJP); wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
37. The wound dressing of claim 36, wherein the functional fragment is a peptide analogue selected from the group consisting of: Ac-LVK-OH (SEQ ID NO: 48), Ac- LIR-OH (SEQ ID NO: 51), Ac-FDR-OH (SEQ ID NO: 53), Ac-HNIR-OH (SEQ ID NO: 59), H-FTK-OH (SEQ ID NO: 88), and Ac-QNGNK-OH (SEQ ID NO: 106).
38. A method of producing an anti-inflammatory molecule that is an apalbumin protein or functional fragment thereof by modifying royal jelly, the method including the step of reacting royal jelly with at least 0.1% MGO at between 18 and 37 degrees Celsius.
39. The method of claim 38 including the step of reacting the royal jelly with at least 0.5% MGO at between 18 and 37 degrees Celsius.
40. The method of claim 38 or claim 39 including the step of reacting royal jelly with at least 1.0% MGO at between 18 and 37 degrees Celsius.
41. A method of identifying (i) the anti-inflammatory capacity or (ii) MGO- modified major royal jelly protein concentration of a sample of honey, comprising the step of: assaying the Cathepsin B inhibition levels of the honey sample.
42. The method as claimed in claim 41, wherein the honey is derived from Leptospermum, including manuka honey or jelly bush honey.
43. The method of claim 41 or claim 42, wherein the method is used to enable a bee keeper to determine the right time to harvest honey from a hive in order to obtain a honey sample containing a desired anti-inflammatory capacity or MGO-modified major royal jelly protein content.
44. The method of claim 41 or claim 42, wherein the method is used to enable a honey producer to determine a desired length of time to store honey, in order to obtain a honey sample with a desired anti-inflammatory capacity and MGO-modified major royal jelly protein content.
45. Use of a functional fragment of a major royal jelly protein (MRJP) for preparing a medicament for inhibiting Cathepsin B activity in a cellular tissue in a patient; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises 2 to 20 amino acids of the last 20 amino acids at the C-terminus of the protein; and wherein a lysine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
46. The use of claim 45, wherein the functional fragment comprises 2 to 10 amino acids of the last 10 amino acids at the C-terminus of the protein.
47. The use of claim 45 or claim 46, wherein the major royal jelly protein (MRJP) is selected from the group consisting of MRJP1 (SEQ ID NO: 1), MRJP2 (SEQ ID NO: 2), MRJP3 (SEQ ID NO: 3), MRJP4 (SEQ ID NO: 4), MRJP5 (SEQ ID NO: 5), MRJP6 (SEQ ID NO: 6), MRJP7 (SEQ ID NO: 7), MRJP8 (SEQ ID NO: 8), and MRJP9 (SEQ ID NO: 9).
48. The use of any one of claims 45 to 47, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. KISIHL (SEQ ID NO: 10); ii. KNNNQNDN (SEQ ID NO: 11); iii. KLH (SEQ ID NO: 12); iv. KSNNRHNNND (SEQ ID NO: 13); v. KHNN (SEQ ID NO: 14); vi. KNQAHLD (SEQ ID NO: 15); vii. KNTRCISP (SEQ ID NO: 16); and viii. KTNFFSIFL (SEQ ID NO: 17).
49. The use of any one of claims 45 to 48, wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: i. PFKISIHL (SEQ ID NO: 64); ii. NQKNNNQNDN (SEQ ID NO: 69); NQKN (SEQ ID NO: 66); iii. SKLH (SEQ ID NO: 71); iv. QNKHNN (SEQ ID NO: 81); and KH (SEQ ID NO: 80).
50. The use of any one of claims 45 to 47, or 49, wherein the functional fragment is a peptide analogue selected from the group consisting of: i. Ac-PFKISIHL-OH (SEQ ID NO: 19); ii. Ac-NQKNNNQNDN-OH (SEQ ID NO: 24); Ac-NQKN-OH (SEQ ID NO: 27); iii. Ac-SKLH-OH (SEQ ID NO: 31); iv. Ac-QNKHNN-OH (SEQ ID NO: 43); and Ac-KH-OH (SEQ ID NO: 45).
51. Use of a functional fragment of a major royal jelly protein (MRJP) for preparing a medicament for inhibiting Cathepsin B activity in a cellular tissue in a patient; wherein the functional fragment has been isolated, enriched, synthesized, or recombinantly produced; wherein the functional fragment comprises an amino acid sequence selected from the group consisting of: LVK (SEQ ID NO: 84), LIR (SEQ ID NO: 86), FDR (SEQ ID NO: 127), HNIR (SEQ ID NO: 128), FTK (SEQ ID NO: 130), and QNGNK (SEQ ID NO: 137); and wherein a lysine or arginine amino acid residue of the functional fragment has been chemically modified by methylglyoxal (MGO).
52. The use of claim 51, wherein the functional fragment is a peptide analogue selected from the group consisting of: Ac-LVK-OH (SEQ ID NO: 48), Ac-LIR-OH (SEQ ID NO: 51), Ac-FDR-OH (SEQ ID NO: 53), Ac-HNIR-OH (SEQ ID NO: 59), H- FTK-OH (SEQ ID NO: 88), and Ac-QNGNK-OH (SEQ ID NO: 106).
53. A method of enriching the anti-inflammatory molecules in a Leptospermum genus derived MGO containing honey comprising the step of adding major royal jelly protein to the honey.
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