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PubMed2005

The genetic epidemiology of human primary osteoarthritis: current status.

Osteoarthritis (OA) is a common disease characterised by the degeneration of the cartilage of synovial joints such as the hip and knee. In the past ten years a large number of twin-pair, sibling-risk and segregation studies have been conducted on the disease, and these have revealed a major genetic component that is transmitted in a nonmendelian manner. OA therefore fits best into the complex, multifactorial class of common diseases. With a genetic component established, genome-wide linkage scans were performed, and these uncovered several genomic intervals likely to harbour OA susceptibility. In the past few years these intervals have started to yield genes containing OA-associated variants. This is therefore a very exciting period in the molecular genetic analysis of this common disease. The genes that have so far been implicated in susceptibility include the interleukin 1 gene (IL1) cluster at chromosome 2q11.2-q13, the matrilin 3 gene (MATN3) at 2p24.1, the IL-4 receptor alpha-chain gene (IL4R) at 16p12.1, the secreted frizzled-related protein 3 gene (FRZB) at 2q32.1, the metalloproteinase gene ADAM12 at 10q26.2 and, most recently, the asporin gene (ASPN) at 9q22.31. The evidence for involvement of these genes in OA is more compelling for some than others, with the IL1 and ASPN associations being the most convincing to date. It is imperative that the veracity of each of the associations be tested by genotyping additional cohorts and that their global relevance be assessed by genotyping OA cohorts from different ethnic backgrounds. The gene products of IL1, IL4R, FRZB and ASPN regulate cartilage chondrocyte differentiation and survival, and their effects on the chondrocyte are potentially amenable to therapeutic intervention. The latest genetics is therefore providing new insights for the development of novel OA treatments.

Cell Differentiation↗
PubMed1993

The role of primary care clinician attitudes, beliefs, and training in the diagnosis and treatment of depression. A report from the Ambulatory Sentinel Practice Network Inc.

OBJECTIVE: To examine the relation between primary care clinicians' attitudes, beliefs, and training and their perceptions of the importance and frequency of depression. DESIGN: A 56-item questionnaire was mailed to 226 clinicians in the Ambulatory Sentinel Practice Network Inc (ASPN), a primary care research network. SETTING: The ASPN, consisting of 69 primary care practices in the United States and Canada, cares for approximately 350,000 patients. MAIN OUTCOME MEASURE: The degree to which clinician training in depression and clinician attitudes and beliefs about treating depression predict their perceptions of the importance of depression in their primary care practices. RESULTS: Results of path analysis indicate that clinician training in depression, beliefs about the burden and discomfort associated with diagnosing and treating depression, perceptions of their patients' discomfort, and self-efficacy in diagnosing and treating depression are all significantly related to clinician perceptions of whether depression was an important and frequent primary care problem. Further analysis indicates that actual prevalence of depression accounted for little variability in clinicians' beliefs and attitudes about depression. CONCLUSIONS: What clinicians think their patients feel about issues on depression and how they view their own abilities may profoundly influence the degree to which they recognize depression in primary care. Understanding these factors may help explain and subsequently decrease clinician variability in the recognition of depression in primary care.

Attitude of Health Personnel↗
PubMed2005

An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis.

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-beta-mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-beta activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-beta. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-beta activity and disease, suggesting new therapeutic strategies for osteoarthritis.

Aggrecans↗
PubMed2006

Serine and cysteine proteases are translocated to similar extents upon formation of covalent complexes with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of the protease binding site in CrmA complexes with granzyme B and caspase-1.

CrmA is a "cross-class" serpin family inhibitor of the proapoptotic serine protease, granzyme B, as well as cysteine proteases of the caspase family. To determine whether crmA inhibits these structurally diverse proteases by a common conformational trapping mechanism, we mapped the position of the protease in crmA complexes with granzyme B or caspase-1 by fluorescence perturbation and fluorescence resonance energy transfer (FRET) analyses of site-specific fluorophore-labeled crmAs. A reactive loop P6 NBD label underwent similar large fluorescence enhancements (>200%) either upon reactive loop cleavage by AspN protease or complex formation with granzyme B or caspase-1, consistent with the insertion of the cleaved reactive loop into sheet A in both types of crmA-protease complexes. NBD labels on the noninserting part of the reactive loop docking site for protease (P1' residue) or midway between the two ends of sheet A (helix F residue 101) showed no significant perturbations due to protease complexation. By contrast, labels at positions 68 and 261, lying at the end of sheet A most distal from the reactive loop, showed marked perturbations distinct from those induced by AspN cleavage and thus ascribable to granzyme B or caspase-1 proximity in the complexes. Substantial FRET between protease tryptophans and 5-dimethylaminonaphthalene-1-sulfonyl-labeled crmAs occurred in protease complexes with crmAs labeled at the 68 and 261 positions, but not the P1' position. These results suggest that granzyme B and caspase-1 are inhibited by crmA by a common mechanism involving full reactive loop insertion into sheet A and translocation of the protease to the distal end of the sheet as previously found for inhibition of other serine proteases by serpins.

Binding Sites↗
PubMed1996

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Amino Acid Sequence↗
PubMed2026

Graph neural network-based risk stratification of prostate cancer using gene expression and SHAP interpretability.

Accurate risk stratification is essential for guiding treatment decisions and preventing over treatment of prostate cancer, which remains one of the most prevalent cancers among adult men. While the Gleason score, obtained from prostate biopsies, is routinely used to assess tumor aggressiveness, the biopsy procedure carries risks such as pain, infection, and, in some cases, serious complications such as sepsis. In this study, we proposed an artificial intelligence-based framework that integrates mRNA expression profiles with functional interaction networks to classify prostate cancer patients into low-, medium-, and high-risk groups defined by Gleason scores. The pipeline comprised five steps: (1) data collection from The Cancer Genome Atlas (TCGA), (2) preprocessing of gene expression data, (3) two-stage feature selection to identify informative biomarkers, (4) risk classification using a dual-branch graph neural network (GNN) that combines gene-gene interaction graphs with sample-level expression features, and (5) model interpretation using SHAP to quantify feature contributions. Differentially expressed genes were identified in the High (ASPN, GMNN, PEBP4, C2, KNCK17), Medium (C2, IGSF1, ASPN, CDKN3, AMH), and Low (TNMD, VWA5B2, ST6GALNAC5, CYP3A5, PHGR1) risk groups, underscoring the molecular heterogeneity of disease progression. On an independent held-out test set, the model achieved AUCs of 0.86, 0.88, and 0.95 for the low-, medium-, and high-risk groups, respectively, with an overall accuracy of 80%. These results suggest that combining GNN-based modeling with explainable AI can capture both global and local molecular patterns relevant to tumor aggressiveness. However, as the model was developed and evaluated solely on the TCGA cohort, the findings should be regarded as exploratory, and external validation will be required to establish generalizability. Within these limitations, the proposed framework highlights the potential of molecular profiling and graph-based deep learning to support more precise, potentially less invasive, risk assessment and individualized treatment planning in prostate cancer.

Prostatic Neoplasms↗
PubMed1992

Patients presenting to family physicians after a fall: a report from the Ambulatory Sentinel Practice Network.

BACKGROUND: Patients who fall present a diagnostic challenge to family physicians. The diagnostic workup of these patients must be thorough enough to detect and treat important causes of the fall yet not subject patients to unnecessary tests. Previous studies have provided only limited guidance for primary care physicians because in general they occurred in settings other than primary care and focused on a single age group. METHODS: The Ambulatory Sentinel Practice Network (ASPN) conducted a 6-month study of primary care patients of all ages presenting after a fall, or with medical problems resulting from a fall. ASPN clinicians collected information about the history, physical examination findings, and follow-up of these patients. Causes of falls were grouped into three categories: external reasons for falling, internal reasons related to gait, and internal reasons unrelated to gait. RESULTS: Participating clinicians identified 431 patients who had falls out of the 256,680 seen for any reason during the study period. The patients ranged in age from 1 to 94 years. The rate of falls for patients increased rapidly after age 65 years. Most falls occurred for reasons external to the patient, but internal reasons, both nonlocomotor and locomotor, increased after age 65 years. No nonlocomotor causes for a fall were found in patients younger than 65 years of age. Also, the rate of hospitalization of patients seen for falls was greater in the geriatric age group. CONCLUSIONS: The results highlight the need for further research about falls, particularly those occurring in pediatric and young adult patients. Furthermore, correcting environmental hazards and modifying gait problems in the elderly by increasing lower extremity and truncal strength could decrease the risk of falling.

Accidental Falls↗
PubMed2006

[Asporin, a susceptibility gene for osteoarthritis].

Osteoarthritis (OA) is the most common form of human arthritis. Genetic factors have been implicated in OA. Through a candidate gene-association study, we found association between asporin and OA. Asporin is an extracellular matrix component expressed abundantly in the articular cartilage of OA patients. A significant association between a polymorphism in the aspartic acid (D) repeat of the asporin gene (ASPN) and knee OA was found; the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in patients with hip OA. The association of asporin is replicated in European population by meta-analysis. Asporin suppresses transforming growth factor-beta (TGF-beta)-mediated expression of aggrecan and type II collagen genes and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. Asporin co-localized to and bound to TGF-beta, and inhibited TGF-beta-Smad signal. Clarification of molecular pathway of OA relating to asporin and TGF-beta would lead to order-made medicine and novel therapeutic strategies for OA.

Alleles↗
PubMedDate not supplied

Pelvic inflammatory disease in primary care.

The Ambulatory Sentinel Practice Network (ASPN) conducted an observational study of pelvic inflammatory disease (PID) in the primary care setting. During 14 months from 1982 to 1983, 38 practices in 16 states and two Canadian provinces reported 384 first visits for patients with PID. PID, as diagnosed by the clinicians in ASPN, was less severe than other published clinical descriptions of PID. Fewer patients had fever, palpable adnexal masses, and extensive tenderness than reported in other series. Although 43% of patients met published recommendations for hospitalization, the clinicians hospitalized only 9% on the initial visit, similar to the hospitalization rate by office gynecologists in a national study. Whether this management represented optimal care in the primary care setting is unknown. If office based physicians in the United States admitted to the hospital all PID patients who met current recommendations for hospitalization, the added annual cost could exceed $1.2 billion. Given both the serious clinical consequences of PID and the enormous financial implications of different clinical strategies, there is a compelling need to investigate the diagnosis and management of PID in primary care.

Adult↗
PubMed1984

The Ambulatory Sentinel Practice Network: purpose, methods, and policies.

The Ambulatory Sentinel Practice Network (ASPN) is a network of primary health care practices across the United States and Canada offering (1) a laboratory for the study of populations under the care of primary care providers, and (2) surveillance of primary care problems and services. This paper reports the methods and policies developed and used by ASPN to conduct studies and describes the initial sentinel practices.

Adolescent↗
PubMed1990

Role of mass spectrometry in mapping strain variation and post-translational modifications of viral proteins.

Enzymatically derived fragments of the nucleocapsid protein from one strain (V4) of the paramyxovirus, New castle disease virus (NDV), have been aligned with the sequence deduced for a related strain (D26) by gene sequence analysis. This process involved extensive use of fast atom bombardment (FAB) mass spectrometry of unfractionated tryptic digests and fragments separated from tryptic or AspN protease digests by high-performance liquid chromatography (HPLC). Amino acid analysis and stepwise Edman degradation sequence analysis were used to complement FAB mass spectral data or as alternatives where no ions were produced by FAB. The nature of biosynthetic processing and blockage (acetylation) at the N-terminus of the protein were confirmed using collision-induced dissociation. Data obtained by direct analysis of the V4 nucleocapsid protein facilitated mapping of sequence variations within the nucleocapsid protein of the antigenically distinct WA2116 strain of NDV. Most of the WA2116 protein was mapped by FAB mass spectrometric analysis of HPLC fractions, thus amino acid analysis or stepwise sequence analysis were only required where FAB mass spectral data were inconclusive or indicated amino acid variations. This approach to comparison of NDV nucleocapsid proteins is proposed as a general strategy for mapping strain variation and post-translational modifications of viral proteins.

Amino Acid Sequence↗
PubMed1995

Prediction of polyelectrolyte polypeptide structures using Monte Carlo conformational search methods with implicit solvation modeling.

Many interesting proteins possess defined sequence stretches containing negatively charged amino acids. At present, experimental methods (X-ray crystallography, NMR) have failed to provide structural data for many of these sequence domains. We have applied the dihedral probability grid-Monte Carlo (DPG-MC) conformational search algorithm to a series of N- and C-capped polyelectrolyte peptides, (Glu)20, (Asp)20, (PSer)20, and (PSer-Asp)10, that represent polyanionic regions in a number of important proteins, such as parathymosin, calsequestrin, the sodium channel protein, and the acidic biomineralization proteins. The atomic charges were estimated from charge equilibration and the valence and van der Waals parameters are from DREIDING. Solvation of the carboxylate and phosphate groups was treated using sodium counterions for each charged side chain (one Na+ for COO-; two Na for CO(PO3)-2) plus a distance-dependent (shielded) dielectric constant, epsilon = epsilon 0 R, to simulate solvent water. The structures of these polyelectrolyte polypeptides were obtained by the DPG-MC conformational search with epsilon 0 = 10, followed by calculation of solvation energies for the lowest energy conformers using the protein dipole-Langevin dipole method of Warshel. These calculations predict a correlation between amino acid sequence and global folded conformational minima: 1. Poly-L-Glu20, our structural benchmark, exhibited a preference for right-handed alpha-helix (47% helicity), which approximates experimental observations of 55-60% helicity in solution. 2. For Asp- and PSer-containing sequences, all conformers exhibited a low preference for right-handed alpha-helix formation (< or = 10%), but a significant percentage (approximately 20% or greater) of beta-strand and beta-turn dihedrals were found in all three sequence cases: (1) Aspn forms supercoil conformers, with a 2:1:1 ratio of beta-turn:beta-strand:alpha-helix dihedral angles; (2) PSer20 features a nearly 1:1 ratio of beta-turn:beta-sheet dihedral preferences, with very little preference for alpha-helical structure, and possesses short regions of strand and turn combinations that give rise to a collapsed bend or hairpin structure; (3) (PSer-Asp)10 features a 3:2:1 ratio of beta-sheet:beta-turn:alpha-helix and gives rise to a superturn or C-shaped structure.

Algorithms↗
PubMed2003

Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry.

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.

Animals↗
PubMed2000

Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis.

VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein.

Acetylation↗
PubMed2026

Exploration and experimental verification of triaptosis-related prognostic genes and cells in gastric cancer.

BACKGROUND: Triaptosis is a recently characterized form of programmed cell death with unclear implications in cancer. This study aimed to investigate the prognostic significance and biological relevance of triaptosis in gastric cancer (GC). METHODS: Transcriptomic and clinical data from TCGA-STAD and GSE62254, and single-cell RNA sequencing data from GSE183904 were analyzed. Triaptosis-related gene (TRG) scores were calculated using single-sample gene set enrichment analysis. Differentially expressed genes identified in TRG-score and GC-versus-normal comparisons underwent functional enrichment, Cox regression, and least absolute shrinkage and selection operator regression to develop an externally validated signature. Immune profiles, pathway activity, somatic mutations, tumor mutational burden (TMB), predicted drug sensitivity, and clinical features were compared by risk group. Single-cell analyses assessed TRG activity, prognostic gene expression, cell-cell communication, and pseudotime. Reverse transcription-quantitative PCR and Western blotting assessed mRNA expression and protein levels, respectively. RESULTS: A TRG-based prognostic model comprising ASPN, GRB14, and VTN was developed and externally validated, effectively distinguishing patients into two distinct risk groups with notably different survival outcomes. mRNA expression of all three genes and their protein levels were significantly higher in SGC-7901 cells than in GES-1 cells. High-risk patients had higher stromal scores and distinct immune profiles; 15 immune cell types differed between groups. Single-cell analysis revealed fibroblasts and pericytes among high-TRG-active cell types. Prognostic genes were significantly overexpressed in fibroblasts, which also showed high TRG activity. Fibroblasts demonstrated enhanced communication with pericytes, whereas tumor-derived fibroblasts showed weaker communication with macrophages, indicating immune microenvironment remodeling. CONCLUSION: The three-gene prognostic signature predicted GC prognosis and was associated with distinct immune and genomic features, suggesting potential value for risk stratification and personalized treatment.

Humans↗
PubMed1993

The primary structure of carboxypeptidase S1 from Penicillium janthinellum.

The complete amino acid sequence of carboxypeptidase S1 from Penicillium janthinellium has been determined by N-terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleaved with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu-specific proteinase from B. licheniformis. The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half-cystine residues and one glycosylated asparagine residue. A comparison to other carboxypeptidases shows that the enzyme is homologous to carboxypeptidase-Y and carboxypeptidase-MIII from malt. Specificity and binding of substrates are discussed from a three-dimensional model based on the known structure of carboxypeptidase-Y from Saccharomyces cereviciae and carboxypeptidase II from wheat.

Amino Acid Sequence↗
PubMed1990

Comparison of the positions and efficiency of cleavage activation of fusion protein precursors of virulent and avirulent strains of Newcastle disease virus: insights into the specificities of activating proteases.

The F1- and F2-polypeptide components of in ovo activated fusion proteins of one virulent (AV or Australia-Victoria) strain, one low-virulence (EG or Eaves-Grimes) strain, and two avirulent (V4 or Queensland and WA2116) strains of Newcastle disease virus (NDV) were isolated and subjected to structural analysis. This included complementary application of amino acid analysis, fast atom bombardment-mass spectrometry, and N-terminal sequence analysis to fragments isolated from AspN protease digests of the F2-polypeptides using HPLC. As a result, the complete sequences of the F2-polypeptides were determined, including documentation of glycosylation of asparagine 54. The sequence of the cleavage-activation site of the WA2116 F0-protein was found to be distinctly different from this site in any other NDV F0-protein. Cleavage activation at the C termini of the F2-polypeptide regions was found to have occurred to approximately equivalent extents at arginines 82 and 85 of the AV and EG strains, but was restricted largely to arginine 85 of the V4 strain and completely to arginine 85 of the WA2116 strain. In each case cleavage activation was apparently succeeded by trimming of the basic residues from the newly formed C termini. Immunochemical analysis with antipeptide antisera showed that the extent of cleavage was influenced by amino acids adjacent to these arginines. These data provide insight into the substrate specificities of the enzymes involved in cleavage activation of the fusion protein precursors.

Amino Acid Sequence↗
PubMed1993

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗