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Introduction

Cheese made from cow's milk contains four major cheese proteins: alpha S1, alpha S2, beta and kappa casein, with alpha S1 and beta casein being the most abundant components. Many genetic variants of these four casein genes have been identified in different breeds of cows[1], often associated with various milk and cheese processing parameters (such as coagulation time)[2], or health effects.

Human breast milk is somewhat different in composition - humans do not have a functioning alpha S2 casein gene, and the dominant proteins are beta and kappa casein, rather than alpha S1 and beta. Interestingly, because of intensive research by the dairy industry, far more is known about bovine casein genetic variants ant their consequences than in human.

In addition to the casein genes, we will also express Fam20C, a Golgi kinase responsible for phosphorylating the caseins

Exon-intron structure of bovine milk proteins:

Organization of the casein genes in the human, mouse, rat, and cattle genome:

Alpha-s1 casein (CSN1S1)

Major component of cow's milk, along with beta casein.

α-s1 Casein is the most prevalent form of casein in bovine milk. It has been reported to exhibit antioxidant and radical scavenging properties.1 It has also been reported to be involved in the transport of and casein from the endoplasmic reticulum to the Golgi apparatus. [Chanat, E., J. Cell Sci., 112, 3399-3412 (1999)] [3]

Genetic variants & health effects

A recent review of milk protein nomenclature (Farrell et al., 2004) indicated 8 αs1-CN (A, B, C, D, E, F, G, H) [...] variants in the Bos genus. [4]

CSN1S1 C positively affected milk coagulation [5]

Sequences

• Bovine: UniProt P02662 (A variant)
• Human: UniProt P47710

Bovine alpha-S1-casein is a 214 AA protein, including a 15 AA secretion signal.

Alpha-S1 is a key protein in milk protein allergy, and there is some data available on immunoglobulin E (IgE)-binding epitopes (i.e. antigenic peptides) in different genetic variants (Lisson et al., 2014), but there seems to be a lot of variability from patient to patient, and it is not clear (to me at least) what the health effects are of the different variants.

Until we can figure out the health effects, I think we should focus on CSN1S1*C for its improved coagulation properties:

>gi|195973860|gb|ACG63494.1| alpha S1 casein [Bos taurus]
MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAME
DIKQMEAESISSSEEIVPNSVEQKHIQKDDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKE
GIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSGKTT
MPLW

Human alpha-S1-casein is present at much lower concentration than in bovine milk, and is only 185 AA:

>gi|1345671|sp|P47710.1|CASA1_HUMAN RecName: Full=Alpha-S1-casein
MRLLILTCLVAVALARPKLPLRYPERLQNPSESSEPIPLESREEYMNGMNRQRNILREKQTDEIKDTRNE
STQNCVVAEPEKMESSISSSSEEMSLSKCAEQFCRLNEYNQLQLQAAHAQEQIRRMNENSHVQVPFQQLN
QLAAYPYAVWYYPQIMQYVPFPPFSDISNPTAHENYEKNNVMLQW

Alignment between the bovine reference sequence (A variant), bovine C variant, and human alpha-S1-casein (only 33% sequence identity):

P02662    1    MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELS--------------KDIGSESTED  66
ACG63494  1    MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELS--------------KDIGSESTED  66
P47710    1    MRLLILTCLVAVALARPKLPLRY---PERLQNPSESS---EPIPLESREEYMNGMNrqrnilrekqtdeiKDTRNESTQN  74

P02662    67   QAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQ  146
ACG63494  67   QAMEDIKQMEAESISSSEEIVPNSVEQKHIQKDDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQ  146
P47710    75   CVVAEPEKMESSISSSSEEMSLSKCA------------------EQFCRLNEYN--QLQLQAAHAQEQIRRMNENSHVQV  134

P02662    147  KEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPL-W  214
ACG63494  147  KEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSGKTTMPL-W  214
P47710    135  P-----------------FQQLNQLAAYPYAVWYY-PQIMQYVPFPPFSDISNPTAHENYEKNNVMLqW  185

Alpha-s2 casein (CSN1S2)

Genetic variants & health effects

A recent review of milk protein nomenclature (Farrell et al., 2004) indicated [...] 4 αs2-CN (A, B, C, D) [...] variants in the Bos genus. [6]

Proteolytic fragments of α-s2 Casein have been shown to exhibit antibacterial activity. Specifically the 39 amino acid casocidin-1 peptide fragment has been shown to inhibit E. coli and Staph. carnosis growth. [Zucht, H. D., FEBS Lett., 371, 185-188 (1995)] [7]

Sequences

• Bovine: UniProt P02663 (A variant)

Bovine Alpha-s2 casein is a 222 AA protein, including a 15 AA secretion signal peptide.

>sp|P02663|CASA2_BOVIN Alpha-S2-casein OS=Bos taurus GN=CSN1S2 PE=1 SV=2
MKFFIFTCLLAVALAKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVR
NANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIV
LNPWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLK
KISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKVIPYVRYL

In human, the alpha-s2 casein gene is duplicated, but both copies (CSN1S2AP, CSN1S2BP ) are truncated shortly after the signal peptide. Therefore, these loci appear to be a pseudogenes.

Beta casein (CSN2)

Main component of cow's and human milk.

β-Casein and its fragments have been implicated in a number of biological functions. The casoparan peptide has been reported to activate macrophage phagocytosis and peroxide release. Casohypotensin and casoparan may be involved in bradykinin regulation. Casohypotensin has also been shown to be a strong inhibitor of endo-oligopeptidase A, a thiol-activated protease capable of degrading bradykinin and neurotensin, and hydrolyzing enkephalin-containing peptides to produce enkephalins. β-Caseins are also a source of casomorphin peptides which exhibit opioid activity binding to opioid receptors. Casomorphins may be the hydrolysis product of dipeptidyl peptidase IV. [Miyamoto, Y., et al., Am. J. Physiol. Renal. Physiol., 252, 670-F677 (1987)] [8]

Genetic variants & health effects

A recent review of milk protein nomenclature (Farrell et al., 2004) indicated [...] 12 β-CN (A1, A2, A3, B, C, D, E, F, G, H1, H2, I) [...] variants in the Bos genus. [9]

"CSN2 B positively affected milk coagulation, whereas CSN2 A(2), in particular, had a negative effect" [10]

"Populations, which consume milk containing high levels of β-casein A2 variant, have a lower incidence of cardiovascular disease and type 1 diabetes. Furthermore, consumption of milk with the A2 variant may be associated with less severe symptoms of autism and schizophrenia." [11] "Human milk, goat milk, sheep milk and other species’ milk contain beta-casein which is ‘A2 like’, because they have a proline at the equivalent position in their beta-casein chains." [12].

The health benefits of "A2 milk" (or rather the health risks associated with A1/B beta-casein) are thought to be due to beta-casomorphin 7 (BCM-7), one of a group of degradation peptides with a chain length of 4–11 amino acids (aa), all starting with tyrosine residue in position 60 (Kostyra et al. 2004). The A1 variant (as well as B, C, F, and G) has a histidine rather than Proline in position 67, resulting in more efficient proteolysis at that location by elastase, and a 4-fold higher BCM-7 level in hydrolysed A1 milk vs A2 milk (Kaminski et al, 2007). Human BCM's also show opioid activity (Koch et al., 1985), but may have different health effects due to some amino acid differences.

Sequences

• Bovine: UniProt P02666 (A2 variant)
• Human: UniProt P05814

Bovine beta-casein is a 224 AA protein, including a 15 AA secretion signal. We should try bovine A2 for the reputed health benefits, and B for improved milk coagulation.

A2:

>gi|115660|sp|P02666.2|CASB_BOVIN RecName: Full=Beta-casein
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQT
QSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSL
TLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQE
PVLGPVRGPFPIIV

B:

>gi|83406093|gb|AAI11173.1| CSN2 protein [Bos taurus]
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQT
QSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTERQSL
TLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQE
PVLGPVRGPFPIIV

Human beta-casein is a 226 AA protein, including a 15 AA secretion signal peptide.

>gi|115661|sp|P05814.4|CASB_HUMAN RecName: Full=Beta-casein
MKVLILACLVALALARETIESLSSSEESITEYKQKVEKVKHEDQQQGEDEHQDKIYPSFQPQPLIYPFVE
PIPYGFLPQNILPLAQPAVVLPVPQPEIMEVPKAKDTVYTKGRVMPVLKSPTIPFFDPQIPKLTDLENLH
LPLPLLQPLMQQVPQPIPQTLALPPQPLWSVPQPKVLPIPQQVVPYPQRAVPVQALLLNQELLLNPTHQI
YPVTQPLAPVHNPISV

Alignment between bovine A2, bovine B, and human (55% identity over 72% of the sequence):

P02666    1    MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGP  80
AAI11173  1    MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGP  80
P05814    1    MKVLILACLVALALARET---------IESLSSSEESITEYKQKVEKVKHEDQQQGEDEHQDKIYPSFQPQPLIYPFVEP  71 

P02666    81   IPNS-LPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWM  159
AAI11173  81   IHNS-LPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTERQSLTLTDVENLHLPLPLLQSWM  159
P05814    72   IPYGfLPQNILPLAQ-PAVVLPVPQPEIMEVPKAKDTVYTKGRVMPVLKSPTIPFFDPQIPKLTDLENLHLPLPLLQPLM  150

P02666    160  HQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP------VRGPF-----PIIV  224
AAI11173  160  HQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP------VRGPF-----PIIV  224
P05814    151  QQVPQPIPQTLALPPQPLWSVPQPKVLPIPQQVVPYPQRAVPVQALLLNQELLLNPthqiypVTQPLapvhnPISV  226

Kappa casein (CSN3)

κ-Casein's orientation on the surface of the casein micelle functions as an interface between the hydrophobic interior caseins and the aqueous environment. During clotting of milk, hydrolysis by chymosin or rennin releases the water soluble fragment, para-k-casein and the hydrophobic caseinomacropeptide. [13]

• Kappa-casein on wikipedia
• Bovine kappa-casein precursor gene CSN3
• kappa-casein genes in other mammals

Genetic variants & health effects

A recent review of milk protein nomenclature (Farrell et al., 2004) indicated [...] 11 κ-CN (A, B, C, E, F1, F2, G1, G2, H, I, J) [...] variants in the Bos genus. [14]

• Casoxins A, B and C have opioid antagonist activity. Casoxin C binds to the complement C3a receptors. Casoplatelin inhibits platelet aggregation. [Lawrence K., Creamer, L. K., et al., Journal of Dairy Science, 81, 3004-3012 (1998)] [15]

• The protein polymorphism at position 148-150 led to 2 different ACE inhibiting peptides. The peptide Val-Ser-Pro derived from CSN3*F1 showed the highest IC50 value (21.8) and is also known from maize (Miyoshi et al., 1991). The peptide Ala-Ser-Pro at the same position of the amino acid sequence but derived from the alleles CSN3*B and CSN3*C is not known for ACE inhibitory effects yet and showed an IC50 value of 242.3.

The genetic variation at amino acid position 96–99 produced 2 further ACE inhibitory peptides. In CSN3*C, the peptide Ala-His-His-Pro (IC50 = 847.6) was identified and at the same position the peptide Ala-Cys-His-Pro (IC50 = 360.7) was identified in CSN3*G2. These 2 peptides are not known for ACE inhibitory effects yet.

The dominant genetic variant of CSN3 in German cattle breeds is CSN3*A, whereas the B allele shows a high frequency in German Brown (0.479; Erhardt, 1989). Compared with CSN3*A, the B allele shows association with higher milk protein content and positive effects on coagulation properties and cheese yield (NgKwai-Hang and Grosclaude, 1992). In addition to these positive attributes, the present study shows another reason for preferring milk with the κ-casein allele B, because of the appearance of a new ACE inhibitory peptide at position 148–150 of the protein. [16]

• CSN3 B positively affected milk coagulation [17]

Sequence

• Bovine: UniProt P02668
• Human: UniProt P07498

Bovine kappa-casein is 190 AA of which the first 21 AA form a secretion signal. The bovine reference sequence is Genbank AY380228, RefSeq NP_776719, corresponding to the CSN*A allele.

Because of improved coagulation time, and possible additional health benefits, we should focus on bovine genetic variant CSN3*B instead:

>gi|36988716|gb|AAQ87923.1| kappa casein [Bos taurus]
MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVA
LINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEI
PTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

(secretion signal in bold; caseinomacropeptide in italic)

The main human variant is:

>gi|1705606|sp|P07498.3|CASK_HUMAN RecName: Full=Kappa-casein
MKSFLLVVNALALTLPFLAVEVQNQKQPACHENDERPFYQKTAPYVPMYYVPNSYPYYGTNLYQRRPAIA
INNPYVPRTYYANPAVVRPHAQIPQRQYLPNSHPPTVVRRPNLHPSFIAIPPKKIQDKIIIPTINTIATV
EPTPAPATEPTVDSVVTPEAFSESIITSTPETTTVAVTPPTA

Alignment between bovine A, bovine B, and human (53% sequence identity):

P02668    1    MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYP  80
AAQ87923  1    MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYP  80
P07498    1    -MKSFLLVVNALALTLPFLAVEVQNQKQPACHENDERPFYQKTAPYVPMYYVPNSYPYYGTNLYQRRPAIAINNPYVPRT  79

P02668    81   YYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAV  160
AAQ87923  81   YYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAV  160
P07498    80   YYANPAVVRPHAQIPQRQYLPN--------SHPPTVVRRPNLHPSFIAIPPKKIQDKIIIPTINTIATVEPTPAPATEPT  151

P02668    161  ESTVATLEDSPEVIE-SPPEINTVQVTSTAV  190
AAQ87923  161  ESTVATLEASPEVIE-SPPEINTVQVTSTAV  190
P07498    152  VDSVVTPEAFSESIItSTPETTTVAVTPPTA  182

Caseinomacropeptide

Kim YJ, Oh YK, Kang W, Lee EY, Park S. Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris. J Ind Microbiol Biotechnol. 2005 Sep;32(9):402-8.

“Caseinomacropeptide is a polypeptide of 64 amino acid residues (106-169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an alpha-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.” “Escherichia coli XL1-Blue (Stratagene, USA) was used for cloning and propagating genes. Host strains for recombinant hCMP were S. cerevisiae 2805 (his-, ura-) [...] For S. cerevisiae, pYIGP (containing a constitutive GAP promoter) or pYEGa (containing a galactose-inducible GAL promoter) was used.”

Role of phosphorylation and glycosylation in micelle formation or coagulation

2-Dimensional electrophoresis (2-DE) can be used to distinguish κ-CN isoforms. In a study of milk from Holstein-Friesian and Jersey cows, about 1/3 of kappa-casein was glycosylated, and almost all phosphorylated. The dominant isoform was 1P (40-50%), with 2P, 1P+1G, 1P+2G and 1P+3G each around 10-15%. [18]

We should investigate if any of this is important for micelle formation or micellar flocculation.

studies on the relationship between κ-CN glycosylation and MCP [Milk Coagulation Properties] provide inconsistent results. Cases et al. (2003) estimated a favorable effect of κ-CN deglycosylation on acid coagulation of milk, with deglycosylated milk exhibiting gel formation at greater pH and increased gel stiffness compared with untreated milk. According to Robitaille et al. (1993) and Mariani et al. (1995), κ-CN glycosylation favorably affects both the rate of firming and the firmness of the curd, but its effects on coagulation time are trivial. Jensen et al. (2012b) described favorable effects of the glycosylated κ-CN fraction on rennet coagulation but, in a later study ( Jensen et al., 2012a), detected no significant difference in the κ-CN posttranslational modification pattern between samples exhibiting good or poor coagulation properties. [...]

Results from this study indicate that variation in RCT [Rennet Coagulation Time] attributable to κ-CNtot must be entirely ascribed to the glycosylated component of the protein. [...]

The degree of κ-CN glycosylation is a key determinant of protein hydrophobicity (O’Connell and Fox, 2000) [...] From a theoretical perspective, an increased degree of glycosylation is thus expected to stabilize the micelle structure (Dziuba and Minkiewicz, 1996) and to increase the time needed by chymosin to activate milk coagulation. However, there is no conclusive evidence that κ-CN is a key element for stability of the native caseinate emulsions [...] [19]

Casein Kinase

There are some confusing factors when talking about casein kinase. There are enzymes in most eukaryotes called Casein Kinase 1 and 2. These apparently don't have much to do with casein!

The name casein kinase 2 arose from the fact that casein is a convenient protein substrate that is generally used to assay its activity ... many researchers that work with this enzyme agreed to abandon the reference to casein because it leads to confusion about a possible physiological role for this substrate. The name protein kinase CK2 was recommended. -- Allende 1995

In fact, until 2012 the kinases responsible for casein phosphorylation were unknown. The actual kinase is a secreted kinase called Fam20C (Tagliabracci et al. 2012, Tagliabracci et al. 2013).

Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization [Casein kinase-1 and casein kinase-2] are physiologically unrelated to casein because they are mainly cytosolic and nuclear proteins that would be unlikely to encounter casein in the secretory pathway and have therefore been renamed protein kinase CK1 and protein kinase CK2. A physiological casein kinase activity has been characterized from highly enriched Golgi fractions of lactating mammary gland, liver, brain, and kidney and named Golgi-enriched fraction casein kinase (GEF-CK). The GEF-CK specifically recognizes the consensus S-x-E/pS (where x is any amino acid and E/pS can be Glu or phosphoserine). [...] A peptide substrate reproducing one of the sites phosphorylated in β-casein, KKIEKFQSE-EQQQ (β28-40), is selectively phosphorylated by GEF-CK. [...] Protein immunoblotting of immunoprecipitates from conditioned medium of U2OS cells overexpressing V5-tagged αs1-casein with FLAG-tagged Fam20C revealed a mobility shift in αs1-casein that was absent when αs1-casein was coexpressed with the inactive Fam20C D478A mutant [...] Thus, Fam20C appears to phosphorylate casein in vitro and in mammalian cells. -- Tagliabracci et al. 2012

Sequence

• Bovine: UniProt F1MXQ3 (unreviewed)
• Human: UniProt Q8IXL6

The Human FAM20C protein (also known as DMP4) is 584 AA, including a 22 AA signal peptide:

>gi|327478506|sp|Q8IXL6.2|DMP4_HUMAN RecName: Full=Extracellular serine/threonine protein kinase FAM20C
MKMMLVRRFRVLILMVFLVACALHIALDLLPRLERRGARPSGEPGCSCAQPAAEVAAPGWAQVRGRPGEP
PAASSAAGDAGWPNKHTLRILQDFSSDPSSNLSSHSLEKLPPAAEPAERALRGRDPGALRPHDPAHRPLL
RDPGPRRSESPPGPGGDASLLARLFEHPLYRVAVPPLTEEDVLFNVNSDTRLSPKAAENPDWPHAGAEGA
EFLSPGEAAVDSYPNWLKFHIGINRYELYSRHNPAIEALLHDLSSQRITSVAMKSGGTQLKLIMTFQNYG
QALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRILDFRRVPPVAGRMVNMTKEIRDVTRDKKLW
RTFFISPANNICFYGECSYYCSTEHALCGKPDQIEGSLAAFLPDLSLAKRKTWRNPWRRSYHKRKKAEWE
VDPDYCEEVKQTPPYDSSHRILDVMDMTIFDFLMGNMDRHHYETFEKFGNETFIIHLDNGRGFGKYSHDE
LSILVPLQQCCRIRKSTYLRLQLLAKEEYKLSLLMAESLRGDQVAPVLYQPHLEALDRRLRVVLKAVRDC
VERNGLHSVVDDDLDTEHRAASAR

Parts Table