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Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining.

Smolenski GA, Cursons RT, Hine BC, Wheeler TT - Vet. Res. (2015)

Bottom Line: The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium.Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated.Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms.

View Article: PubMed Central - PubMed

Affiliation: Dairy Foods, AgResearch Ltd, Ruakura Research Centre, Hamilton, 3240, New Zealand. grant.smolenski@agresearch.co.nz.

ABSTRACT
The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis.

No MeSH data available.


Related in: MedlinePlus

2-DE proteome map of bovine teat canal lining and teat skin. (A) Pooled TCL proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The relative molecular weight (MW) is given on the left, while the pI range is given at the top of the figure. Thirty six protein spots corresponding to identified keratin proteins and 17 S100 protein spots (enclosed in the box) are indicated by arrows. Keratin abbreviations are (K1) keratin, type II, cytoskeletal 1; (K3) keratin, type II, cytoskeletal 3; (K4) keratin, type II, cytoskeletal 4; (K5) keratin, type II, cytoskeletal 5; (K6a) keratin, type II, cytoskeletal 6A; (K6c) keratin, type II, cytoskeletal 6C; (K10) keratin, type I, cytoskeletal 10; (K14) keratin, type I, cytoskeletal 14; (K17) keratin, type I, cytoskeletal 17; (K59) keratin, type II, cytoskeletal 59, component IV; (K79) keratin, type II, cytoskeletal 79. (B) Pooled teat skin proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The absence or lowered relative abundance of keratin proteins, compared to TCL, is indicated by dotted circles. Keratin and S100 protein spots identified by MS are indicated by arrows. TCL, teat canal lining; TS, teat skin.
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Fig1: 2-DE proteome map of bovine teat canal lining and teat skin. (A) Pooled TCL proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The relative molecular weight (MW) is given on the left, while the pI range is given at the top of the figure. Thirty six protein spots corresponding to identified keratin proteins and 17 S100 protein spots (enclosed in the box) are indicated by arrows. Keratin abbreviations are (K1) keratin, type II, cytoskeletal 1; (K3) keratin, type II, cytoskeletal 3; (K4) keratin, type II, cytoskeletal 4; (K5) keratin, type II, cytoskeletal 5; (K6a) keratin, type II, cytoskeletal 6A; (K6c) keratin, type II, cytoskeletal 6C; (K10) keratin, type I, cytoskeletal 10; (K14) keratin, type I, cytoskeletal 14; (K17) keratin, type I, cytoskeletal 17; (K59) keratin, type II, cytoskeletal 59, component IV; (K79) keratin, type II, cytoskeletal 79. (B) Pooled teat skin proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The absence or lowered relative abundance of keratin proteins, compared to TCL, is indicated by dotted circles. Keratin and S100 protein spots identified by MS are indicated by arrows. TCL, teat canal lining; TS, teat skin.

Mentions: Analysis of pooled teat canal lining by 2-DE revealed a complex pattern of spots dominated by two clusters of proteins of relatively high abundance (Figure 1A). The main cluster contained many resolvable protein spots between 50 to 70 kDa ranging from pI 4.5 to 9.5. These were estimated to comprise 51% of the total resolvable protein by densitometry. A total of 36 of these spots were identified by MS as keratins (indicated by arrows labelled with a K in Figure 1A). Some of the complexity observed among the keratins is likely due to variable phosphorylation as has been previously reported to be the case for keratins in other epithelial tissues [23]. The primary (cell-type defining) keratins K1 [GenBank: XP_003584943], K3 [XP_003584947], K4 [NP_001091855], K5 [DAA30012], K10 [NP_776802] and K14 [NP_001160047] were identified as well as the secondary keratins K6a [NP_001076979], K6c [XP_884760], K17 [A1L595], K59 [NP_001244333] and K79 [NP_001068816]. Two different isoforms of keratin K6 were identified with the K6a isoform predominating over the K6c isoform. This mixture of keratins is consistent with proteins originating from hyper-proliferative stratified squamous epithelium.Figure 1


Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining.

Smolenski GA, Cursons RT, Hine BC, Wheeler TT - Vet. Res. (2015)

2-DE proteome map of bovine teat canal lining and teat skin. (A) Pooled TCL proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The relative molecular weight (MW) is given on the left, while the pI range is given at the top of the figure. Thirty six protein spots corresponding to identified keratin proteins and 17 S100 protein spots (enclosed in the box) are indicated by arrows. Keratin abbreviations are (K1) keratin, type II, cytoskeletal 1; (K3) keratin, type II, cytoskeletal 3; (K4) keratin, type II, cytoskeletal 4; (K5) keratin, type II, cytoskeletal 5; (K6a) keratin, type II, cytoskeletal 6A; (K6c) keratin, type II, cytoskeletal 6C; (K10) keratin, type I, cytoskeletal 10; (K14) keratin, type I, cytoskeletal 14; (K17) keratin, type I, cytoskeletal 17; (K59) keratin, type II, cytoskeletal 59, component IV; (K79) keratin, type II, cytoskeletal 79. (B) Pooled teat skin proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The absence or lowered relative abundance of keratin proteins, compared to TCL, is indicated by dotted circles. Keratin and S100 protein spots identified by MS are indicated by arrows. TCL, teat canal lining; TS, teat skin.
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Related In: Results  -  Collection

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Fig1: 2-DE proteome map of bovine teat canal lining and teat skin. (A) Pooled TCL proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The relative molecular weight (MW) is given on the left, while the pI range is given at the top of the figure. Thirty six protein spots corresponding to identified keratin proteins and 17 S100 protein spots (enclosed in the box) are indicated by arrows. Keratin abbreviations are (K1) keratin, type II, cytoskeletal 1; (K3) keratin, type II, cytoskeletal 3; (K4) keratin, type II, cytoskeletal 4; (K5) keratin, type II, cytoskeletal 5; (K6a) keratin, type II, cytoskeletal 6A; (K6c) keratin, type II, cytoskeletal 6C; (K10) keratin, type I, cytoskeletal 10; (K14) keratin, type I, cytoskeletal 14; (K17) keratin, type I, cytoskeletal 17; (K59) keratin, type II, cytoskeletal 59, component IV; (K79) keratin, type II, cytoskeletal 79. (B) Pooled teat skin proteins (350 μg) were subjected to 2-DE and detected by colloidal Coomassie blue staining. The absence or lowered relative abundance of keratin proteins, compared to TCL, is indicated by dotted circles. Keratin and S100 protein spots identified by MS are indicated by arrows. TCL, teat canal lining; TS, teat skin.
Mentions: Analysis of pooled teat canal lining by 2-DE revealed a complex pattern of spots dominated by two clusters of proteins of relatively high abundance (Figure 1A). The main cluster contained many resolvable protein spots between 50 to 70 kDa ranging from pI 4.5 to 9.5. These were estimated to comprise 51% of the total resolvable protein by densitometry. A total of 36 of these spots were identified by MS as keratins (indicated by arrows labelled with a K in Figure 1A). Some of the complexity observed among the keratins is likely due to variable phosphorylation as has been previously reported to be the case for keratins in other epithelial tissues [23]. The primary (cell-type defining) keratins K1 [GenBank: XP_003584943], K3 [XP_003584947], K4 [NP_001091855], K5 [DAA30012], K10 [NP_776802] and K14 [NP_001160047] were identified as well as the secondary keratins K6a [NP_001076979], K6c [XP_884760], K17 [A1L595], K59 [NP_001244333] and K79 [NP_001068816]. Two different isoforms of keratin K6 were identified with the K6a isoform predominating over the K6c isoform. This mixture of keratins is consistent with proteins originating from hyper-proliferative stratified squamous epithelium.Figure 1

Bottom Line: The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium.Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated.Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms.

View Article: PubMed Central - PubMed

Affiliation: Dairy Foods, AgResearch Ltd, Ruakura Research Centre, Hamilton, 3240, New Zealand. grant.smolenski@agresearch.co.nz.

ABSTRACT
The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis.

No MeSH data available.


Related in: MedlinePlus