It has recently been shown that the fat-derived hormone adiponectin has the ability to decrease hyperglycemia and to reverse insulin resistance. However, bacterially produced full-length adiponectin is functionally inactive. Here, we show that endogenous adiponectin secreted by adipocytes is post-translationally modified into eight different isoforms, as shown by two-dimensional gel electrophoresis. Carbohydrate detection revealed that six of the adiponectin isoforms are glycosylated. The glycosylation sites were mapped to several lysines (residues 68, 71, 80, and 104) located in the collagenous domain of adiponectin, each having the surrounding motif of GXKGE(D). These four lysines were found to be hydroxylated and subsequently glycosylated. The glycosides attached to each of these four hydroxylated lysines are possibly glucosylgalactosyl groups. Functional analysis revealed that full-length adiponectin produced by mammalian cells is much more potent than bacterially generated adiponectin in enhancing the ability of subphysiological concentrations of insulin to inhibit gluconeogenesis in primary rat hepatocytes, whereas this insulin-sensitizing ability was significantly attenuated when the four glycosylated lysines were substituted with arginines. These results indicate that full-length adiponectin produced by mammalian cells is functionally active as an insulin sensitizer and that hydroxylation and glycosylation of the four lysines in the collagenous domain might contribute to this activity.In addition to serving as an energy storage depot for triglycerides, adipose tissue is now recognized as an active endocrine organ that can secret a variety of biologically active molecules (adipocytokines) in response to extracellular signals (1-4). Some of these adipocytokines, such as leptin, tumor necrosis factor-␣, and resistin, have been shown to play critical roles in the regulation of systemic energy homeostasis, and altered expression and/or secretion of these adipocytokines may contribute to the causation of insulin resistance, type II diabetes, and its complications such as cardiovascular diseases.Adiponectin (also called ACRP30, adipoQ, and GBP28) is a protein exclusively secreted by adipocytes and was originally cloned by four research groups using different approaches (5-8). Several recent studies suggest that adiponectin might be a critical, long sought after hormone that links obesity, insulin resistance, and type II diabetes (9 -11). The adiponectin gene is located in chromosome 3q27, a susceptibility locus for type II diabetes and related metabolic syndromes (12-14). Circulating adiponectin is abundant in humans as well as rodents, with plasma circulating levels in the microgram/ml range, accounting for ϳ0.01% of the total plasma protein (15, 16). mRNA expression and the secretion level of adiponectin are dramatically decreased in a variety of animal models of insulin resistance as well as in obese humans and type II diabetic patients from different ethnic groups (15)(16)(17)(18)(19)(20). Notably, treatmen...

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl-and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.Mucins are high-molecular-weight glycoproteins which form the structural component of the protective mucus gel layer at the surfaces of the gastrointestinal, respiratory, and female genital tracts. Colonic mucin, particularly in the proximal region, contains significant levels of sulfate covalently bound to the mucin oligosaccharide chains, and levels of 2.0 to 6.5 g of sulfate per 100 g of mucin have been found (9, 17). Heavily sulfated mucins (sulfomucins) have many of the general lubricating and barrier functions of mucins with lower sulfate levels. There is accumulating evidence that sulfomucins may, in addition, rate-limit mucin degradation by mucin-degrading bacterial enzymes (4,7,13,15,18,24,26,27), and this role is thought to be particularly important in the colon, where approximately 10 14 bacterial cells are located (10). There have been reports of mucin-desulfating sulfatases that partially remove the sulfate from sulfomucin in a number of bacteria from the mouth, stomach, and colon and in feces. The effect of such sulfatases is to increase the susceptibility of the mucin to degradation by other mucin-degrading enzymes (4, 27). Elevated levels of bacterial mucin-desulfating sulfatases are found in feces of patients with ulcerative colitis (26). The sulfated sugar specificity of these fecal sulfatases, the number of different types present, the bacterial origin of the elevated levels, and the conditions which regulate bacterial production of such enzymes are unknown. The types of mucin-desulfating sulfatases that might be predicted include sulfatases specific for galactose-3-sulfate, g...

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.

The proteins expressed by a genome have been termed the proteome. By comparing the proteome of a disease‐affected tissue with the proteome of an unaffected tissue it is possible to identify proteins that play a role in a disease process. The hippocampus is involved in the processing of short‐term memory and is affected in Alzheimer's disease. Any comparative proteome analysis that can identify proteins important in a disease affecting the hippocampus requires the characterization of the normal hippocampal proteome. Therefore, we homogenised normal hippocampal tissue and separated the proteins by two‐dimensional polyacrylamide gel electrophoresis (2DE). Seventy‐two unique protein spots were collected from Coomassie blue‐stained 2DE gels and subjected to in‐gel digestion with trypsin, reversed‐phase high‐pressure liquid chromatography peptide separation, and N‐terminal protein sequencing. Sufficient protein sequence was obtained to successfully characterize 66 of the 72 protein spots chosen (92%). Three of the 66 proteins were not present in any database (4.5%). The characterized proteins comprised two dominant functional groups, i.e., enzymes involved in intermediary cellular metabolism (40%), and proteins associated with the cytoskeleton (15%). The identity, molecular mass, isoelectric point, and relative concentration of the characterized proteins are described and constitute a partial proteome map of the normal human hippocampus. Hippocampus 1999;9:644–650. © 1999 Wiley‐Liss, Inc.