Skeletal muscle is an heterogeneous tissue with various biochemical and physical properties of several fiber types. In this study, we carried out the comparative study of protein expression patterns in white and red muscles using two-dimensional gel electrophoresis (2-DE). From more than 500 protein spots detected on each 2-DE gel, we screened five proteins that were differentially expressed between white and red muscles. Using peptide mass fingerprint and tandem mass spectrometry analysis these proteins were identified as myoglobin, two slow-twitch isoforms of myosin light chain and two small heat shock proteins (HSP20 and HSP27). The protein levels of myoglobin, myosin light chain and HSP20 were higher in red muscle, whereas HSP27 was higher in white muscle. In addition, genes of the identified proteins were cloned and their mRNAs were examined. Positive correlations between protein content and their mRNA levels were observed in white and red muscle. These results may provide us with valuable information to understand the different expression profiling between white and red muscle at the protein level.

BackgroundMarbling (intramuscular fat) is a valuable trait that impacts on meat quality and an important factor determining price of beef in the Korean beef market. Animals that are destined for this high marbling market are fed a high concentrate ration for approximately 30 months in the Korean finishing farms. However, this feeding strategy leads to inefficiencies and excessive fat production. This study aimed to identify candidate genes and pathways associated with intramuscular fat deposition on highly divergent marbling phenotypes in adult Hanwoo cattle.ResultsBovine genome array analysis was conducted to detect differentially expressed genes (DEGs) in m. longissimus with divergent marbling phenotype (marbling score 2 to 7). Three data-processing methods (MAS5.0, GCRMA and RMA) were used to test for differential expression (DE). Statistical analysis identified 21 significant transcripts from at least two data-processing methods (P < 0.01). All 21 differentially expressed genes were validated by real-time PCR. Results showed a high concordance in the gene expression fold change between the microarrays and the real time PCR data. Gene Ontology (GO) and pathway analysis demonstrated that some genes (ADAMTS4, CYP51A and SQLE) over expressed in high marbled animals are involved in a protein catabolic process and a cholesterol biosynthesis process. In addition, pathway analysis also revealed that ADAMTS4 is activated by three regulators (IL-17A, TNFα and TGFβ1). QRT-PCR was used to investigate gene expression of these regulators in muscle with divergent intramuscular fat contents. The results demonstrate that ADAMTS4 and TGFβ1 are associated with increasing marbling fat. An ADAMTS4/TGFβ1 pathway seems to be associated with the phenotypic differences between high and low marbled groups.ConclusionsMarbling differences are possibly a function of complex signaling pathway interactions between muscle and fat. These results suggest that ADAMTS4, which is involved in connective tissue degradation, could play a role in an important biological pathway for building up marbling in cattle. Moreover, ADAMTS4 and TGFβ1could potentially be used as an early biological marker for marbling fat content in the early stages of growth.

This study was conducted to identify marbling-related candidate genes in M. longissimus dorsi of high-and low-marbled Hanwoo. The longissimus dorsi muscles were selected for gene expression from eight Hanwoo steer carcasses based on crude fat content. In the analysis of variance, gene expression of five candidate genes, FABP4, SCD, PPARγ, Titin and Nebulin was determined to be significantly different between high-and low-marbled Hanwoo steers (P ＜ 0.0001). The Pik-4 and CaMK II genes were also shown to have a significant effect on crude fat content (P ＜ 0.01). In the analysis of the differential expression between high-and low marbled groups, FABP4 gene expression was approximately 2 times higher in the high marbled group relative to the low marbled group. However, the PPARγ and SCD gene were highly expressed in the low marbled group. In addition, Titin and Nebulin were highly expressed in the low marbled group when placed under relatively high shear force. Finally, the Pik-4 and CaM K II gene also displayed a high expression pattern in the low marbled group. [BMB reports 2008; 41(12): 846-851]

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In previous proteomic studies, heat shock protein β 1 (HSPB1) was detected as a candidate protein related to meat quality in cattle. This study sought to determine if its gene expression was associated with intramuscular fat content in the longissimus thoracis muscle of Korean cattle (Hanwoo). Tissue from two groups of 10 steers each, low-marbling (mean intramuscular fat content, 7.4 ± 1.5%) and high-marbling (23.5 ± 2.8%), were used for immunoblotting, real-time PCR, and statistical analyses. HSPB1 expression in both mRNA and protein was shown to be negatively related to intramuscular fat content (P < 0.05). Pathway analysis found two genes, TNF receptor superfamily member 6 (FAS) and angiotensinogen (AGT), that were regulators of the HSPB1 gene. The expression of the two genes showed a negative correlation with intramuscular fat content (P < 0.05). These results suggest that HSPB1, FAS, and AGT may be good candidate genes associated with intramuscular fat content in the longissimus muscle of Korean cattle.

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A cDNA clone, PoMTP, encoding a putative metzincin family metalloprotease was isolated from the expressed sequence tags of a basidiomycete Pleurotus ostreatus. The 5'-end sequence of PoMTP was determined by the 5'-RACE method. Full-length cDNA sequence (1140 bp) of PoMTP contained a 870 bp open reading frame encoding a protein product of 290 amino acids in addition to a 99 bp of 5'-untranslated sequence and a 171 bp of 3'-untranslated sequence with a poly(A) tail. The deduced amino-acid sequences of PoMTP contained an extensive zinc-binding consensus sequence and a so-called Met-turn sequence which are typical for the metzincin family of metalloproteases, indicating that the PoMTP protein belongs to the metzincin metalloproteases. Four cysteine residues were also observed in the zinc-binding region of PoMTP amino-acid sequence, which are known to be important for the structure and the function of some subfamilies of the metzincins. Comparison of the PoMTP in sequence database showed no significant homology with functionally known metalloproteases of Armillaria mellea, Grifola frondosa, Lentinula edodes, Pleurotus ostreatus, Schizophyllum commune and Tricholoma saponaceum in mushroom. Northern blot and qunatitative RT-PCR analyses indicated the PoMTP mRNA to be abundant at primordial and fruit body stages, but scarce at the mycelial stage, suggesting that the PoMTP metalloprotease plays an important role in mushroom fruiting.

Two genetically different pig breeds, the Korean native pig (KNP) and the Western meat-producing Landrace, show breed-specific traits in stress responsiveness (stress hormone levels), growth performance (live weight), and meat quality (intramuscular fat content). We analyzed expression levels within the proteome and transcriptome of the longissimus muscles of both breeds using two-dimensional electrophoresis (2-DE) and microarray analysis. We constructed a porcine proteome database focused mainly on mitochondrial proteins. In total, 101 proteins were identified, of which approximately 60% were metabolic enzymes and mitochondrial proteins. We screened several proteins and genes related to stress and metabolism in skeletal muscles using comparative analysis. In particular, three stress-related genes (heat shock protein beta-1, stress-70 protein, and heat shock 70 kDa protein) were more highly expressed in the Landrace than in the KNP breed. Six metabolism-related genes (peroxisome proliferative activated receptor alpha, short-chain acyl-CoA dehydrogenase, succinate dehydrogenase, NADH-ubiquinone oxidoreductase, glycerol-3-phosphate dehydrogenase, and sterol regulatory element binding protein-1c), all of which are involved in energy and lipid metabolism, were more highly expressed at the protein or mRNA level in the KNP breed. These data may reflect the breed dependence of traits such as stress responsiveness, growth performance, and meat quality.