Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Abstract

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month-versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluoresceinconjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

“…Pyruvate isoform PK-M1 exists as 5 sub-species with differing isoelectric points in skeletal muscle tissues, whereby theses isozymes display varying degrees of phosphorylation and glycosylation [21,24]. Previous studies have clearly shown that the PK isoform exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa is several-fold decreased in senescent muscle fibres [21]. Other PK forms with a more acidic or basic pI-value did not show major alterations in concentration levels between crude total protein extracts from young versus aged muscle [18].…”

“…These proteomic profiling studies have focused on crude soluble extracts and have shown that muscle aging is associated with altered expression levels of many key contractile proteins, regulatory elements, ion handling proteins, metabolic enzymes and mediators of the cellular stress response [14][15][16][17][18][19][20]. The proteomic finding of a drastically decreased density and reduced enzymatic activity of certain glycolytic enzymes in senescent fibres [21] is in agreement with the idea of a fast-to-slow transformation process during aging [13]. In analogy, proteomic surveys clearly revealed increased levels of mitochondrial enzymes and metabolite transporters of aerobic metabolism in aged muscle [22].…”

“…Although fast and slow isoforms of myosin heavy chain switch in the majority of aged fibres, the dramatic increase in slow MLC2 expression was restricted to individual senescent cells [23]. Since posttranslational modifications play a central role in protein function and considerably increase protein diversity within the skeletal muscle proteome, it is important to stress that glycosylation, tyrosine nitration, and tyrosine/threonine phosphorylation are generally altered in senescent skeletal muscles [21,24,25], thereby complicating global protein expression analysis.…”

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

“…The proteomic profiling of crude soluble extracts has revealed a severely perturbed protein expression pattern in aged muscles involving enzymes of various metabolic pathways, regulatory components of ion homeostasis and elements of the cellular stress response (Piec et al, 2005;Gelfi et al, 2006;O'Connell et al, 2007;Doran et al, 2008;Lombardi et al, 2009;Capitanio et al, 2009). Altered post-translational modifications affecting tyrosine nitration, tyrosine/threonine phosphorylation and N-glycosylation were recently documented to occur to a large extent during biological aging of skeletal muscle fibres (Kanski et al, 2005;Gannon et al, 2008;O'Connell et al, 2008b). The proteomic finding that the ratelimiting glycolytic enzyme pyruvate kinase is greatly reduced in aged muscle, while the expression of key mitochondrial proteins is increased in sarcopenia, strongly suggests a shift towards a more aerobicoxidative metabolism in senescent muscle fibres.…”

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging

“…Protein profiling of whole tissue homogenates has been performed using twodimensional gel electrophoresis (2DGE) 1 and mass spectrometry to identify the proteins differentially expressed during aging in rat (4 -6) and human muscle (7,8). Other studies have focused on specific fractions such as mitochondrial proteins (9), phosphoproteins (10), glycoproteins (11), basic proteins (12), or calpain interacting proteins (13). The few proteomic studies available on human skeletal muscle are mostly based on the 2DGE approach, which implies focusing on a specific pH range (7,8).…”