FINAL REPORT OF UGC‐MAJOR RESEARCH PROJECT Proteoglycomic analysis of glycoconjugates from buffalo (Bubalus bubalis) colostrum Dr. H. S. Aparna Principal Investigator Professor in Biotechnology Department of Biotechnology, Mysore University, Manasagangotri, Mysore ‐570 005, Karnataka, India. Cell no:+919448178576 Email:[email protected]
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(4.5%), lysosome (4.5%) and golgi apparatus (4.5%) in milk (Figure 2A) while, colostrum
proteins (Figure 2B) were classified as extra cellular (20%), nucleus (13%), cytoplasm (30%),
membrane associated (15.5%), peroxisome (4.5%), mitochondria (15.5%) and lysosome (2.2%).
The protein-protein interaction of MFGM and CFGM proteins was performed using
STRING analysis software. The milk proteins were found to interact with various proteins (7
interactions) but in colostrum only 4 interactions were observed (Figures 3A and B).
Figure 2A. Pie chart representation of identified MFGM proteins in subcellular organelles through YLoc
Figure 2B. Pie chart representation of identified CFGM proteins in subcellular organelles through YLoc
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Figure 3. STRING prediction of associated networks among MFGM (A) & CFGM (B) proteins The node represents the differentially accumulated protein while the different
coloured edges represents different evidences for the predicted functional relationship between proteins: green line- neighbourhood genome evidence; dark blue line- co-occurrence evidence; black line- co- expression evidence; yellow line- text-mining evidence
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3. The release of glycans from the glycoconjugates and their structural analysis
The structural analysis of N- and O-linked and GSL glycans was performed by
chemoselective glycoblotting technique. Quantitation of individual glycans was carried out using
internal standards prior to glycoblotting. The N-linked oligosaccharides released from milk and
colostrum protein backbone after PNGase F treatment were derivatized and analyzed by
MALDI-TOF-MS. A sum total of 54 and 41 N-linked oligosaccharides were identified in
colostrum and milk samples, respectively (Figure 4A and B; Tables 3A and B). On the basis of
their structural complexity, N-linked glycans were classified as sialyl, neutral, and high-mannose
type of oligosaccharides. There were higher number of sialyl glycans in colostrum (34)
compared to milk (21) FGM, while neutral (14) and high- mannose (6) glycans were found to be
the same in both samples. Among sialyl oligosaccharides, mono-, di-, and trisialyl glycans were
present in CFGM, while MFGM was found to contain only mono- and disialyl glycans. In fact,
monosialyl glycans were predominant in both CFGM and MFGM proteins. Many of the glycan
structures showed increasing complexity, either by the addition of fucose on the reducing end
GlcNAc or by the addition of fucose and/or sialic acid on the exposed non-reducing end. Thus,
we unravelled significant structural differences between CFGM and MFGM oligosaccharides.
To further confirm the glycoforms of abundant glycans, TOF/ TOF analysis was performed. The
m/z 1768, 1914, and 2073 of CFGM and 2219, 2260, and 2584 of MFGM were chosen for
analysis.
O-linked oligosaccharides are the major glycan structures from the high molecular weight
glycoproteins from colostrum and milk FGM. It was observed that the O-linked glycan profile of
CFGM was different from that of the MFGM sample. Buffalo MFGM was typically found to
contain only 13 oligosaccharides, while in CFGM there were 16 oligosaccharides (Figure 5A and
B; Tables 4A and B). In both samples predominantly core 1 oligosaccharides were present, and
possible structures of core 2, core 3, and core 4 glycans were noticeable, which needs further
confirmation. The overall observation explicates the differences in their number and type of
sialyl glycans between colostrum and milk samples, wherein, CFGM was found to be rich in
sialylated oligosaccharides that decreased during the course of its maturation to milk.
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The glycan analysis from colostrum and milk fat globule membrane GSL revealed the
variations between the samples (Figure 6A and B; Tables 5A and B). The major gangliosides
were found to be GM3 and GD3 irrespective of the samples, but their proportions were almost 3-
fold higher in colostrum compared to milk.
Figure 4. MALDI-TOF MS spectra of N-linked oligosaccharides from buffalo colostrum FGMP (A) and milk FGMP (B).
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Figure 5. MALDI-TOF MS spectra of O-linked oligosaccharides from buffalo colostrum FGMP (A) and buffalo milk FGMP (B).
Figure 6. MALDI-TOF MS spectra of glycosphingolipids from buffalo colostrum FGM (A) and milk FGM (B).
4. Determination of functional properties of glycoconjugates
The bio-functionality of peptides was studied by in-silico method through Bioranker and
BIOPEP. Majority of the peptides were found to possess antioxidant property. Hence antioxidant
activity for the peptide fractions was carried out including cell based antioxidant assays like
GSH, H2O2, ROS, catalase, LDH, ACP and ALP in the presence of DNP, a known oxidant on
blood components. In ABTS free radical scavenging assay, MFGM and CFGM samples showed
54 & 58%, 66 & 70%, 73 & 78, 81 & 88% scavenging at 250, 500, 750 and 1000 μg of peptides
respectively. The results indicated increased radical scavenging activity with increasing
concentration of peptides. However, CFGM peptides were more effective antioxidants than
MFGM peptides. On the assays such as reduced glutathione, endogenous antioxidant marker
assays and the lysosomal enzyme assay MFGM and CFGM peptide fractions were found to show
protection against oxidant stress, wherein, CFGM peptides were more efficient than MFGM
permeate (Figure 7).
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Figure 7. Antioxidant activity of FGM peptides from Buffalo milk and Colostrum. (A) ABTS (B) GSH (C) H2O2 (D) ROS (E) Catalase (F) LDH (G) ACP (H) ALP. I-control, II-blood treated with DNP, III-blood treated with DNP pre incubated with MFGM peptides, IV-blood treated with DNP pre incubated with CFGM peptides, V-blood treated with MFGM peptides alone, VI-blood treated with CFGM peptides alone. Bars indicate ± SE. Means designated with the same letter are not significantly different according to Tukey's HSD at P < 0.05.
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To verify the antimicrobial potency of both colostrum and milk-derived fat globule
membrane proteins (FGMPs), the minimum inhibitory concentration on human pathogens E.
coli, S. aureus, K. pneumoniae, and P. aeruginosa was tested (Table 6). FGMPs from colostrum
and milk were tested with concentrations ranging from 0.05 to 2 mg/mL. Interestingly, CFGM
N-glycan-enriched sample after trypsin digestion followed by PNGase treatment exhibited
growth inhibition with MIC as low as 128 μg/mL for P. aeruginosa and 256 μg/mL for E. coli, S.
aureus, and K. pneumoniae. However, the MFGM sample after PNGase treatment showed MIC
of 256 and 512 μg/mL for P. aeruginosa and S. aureus, respectively, with no effect on E. coli
and K. pneumoniae.
Table 6. Antimicrobial activity of PNGase-F treated Buffalo Colostrum FGMP and
Milk FGMP
Test sample
Solvent
Bacterial Strains (µg/mL)
Escherichia
coli
MTCC7410
Staphylococcus
aureus
MTCC7443
Klebsiella pneumoniae
MTCC109
Pseudomonas
aeruginosa
ATCC9027
Colostrum DMSO 256 256 256 128
Milk DMSO NI* 512 NI* 256
Chloramphenicol DMSO 4 4 8 38
Values are mean of triplicates. *NI-No inhibition
CONCLUSIONS
The FGM proteins isolated from both buffalo milk and early milk were subjected for in
vitro simulation digestion mimicking the digestive system of human beings. The peptides
derived from both the samples were analyzed by nLC-ESI MS/MS to unravel their sequences
and to match the component proteins of FGM. The peptide libraries created facilitated clustering
of milk and colostrum FGM proteins into various functional groups involved in lipid metabolism
and energy production, protein synthesis and secretion, transport, cell signalling, catalytic and
also in immune functions. The STRING and DAVID analyses revealed interactive protein
networks and subcellular localization in both the samples. The N- and O-glycans were released
along with glycans from GLS of both MFGM and CFGM samples and characterized by MALDI-
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TOF-MS & TOF/TOF analyses. The antibacterial activity confirmed with CFGM N-glycans
explicit their role in conferring innate immunity against pathogenic microbes. The peptides of
milk and colostrum FGM offered cellular protection as powerful antioxidants indicated their
promising perspectives in commercial formulations and nutraceuticals. The peptide and glycan
libraries created for FGM proteins from buffalo milk and colostrum in the present study provide
ample scope to explore newer functionalities for this food based bioactive molecules.
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PUBLICATIONS
1. Comprehensive characterization of bioactive peptides from Buffalo (Bubalus
bubalis) colostrum and milk fat globule membrane proteins
Brijesha N and Aparna H S
Food Research International, 97: 95–103, 2017. (IF- 4.972)
2. A comparative glycomics of fat globule membrane glycoconjugates from Buffalo
(Bubalus bubalis) milk & colostrum
Brijesha N, Nishimura S –I. and Aparna H S
Journal of Agricultural and Food Chemistry 65: 1496-1506, 2017. (IF- 4.192)
3. Buffalo (Bubalus bubalis) colostrum and milk fat globule membrane fractions are
potent antioxidants
Brijesha N and Aparna H S
Journal of Applied Biology & Biotechnology, 5: 89-93, 2017.