Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation.

OBJECTIVES: A collagen degradation activity (CDA) assay was developed to improve the biochemical characterization of purified collagenase used for islet isolation. MATERIALS AND METHODS: Purified class I collagenase (CI) or class II collagenase (CII) from Clostridium histolyticum cultures were used in all experiments. The CDA assay was performed by incubating 50 microg/mL of FITC fibrils with CI or CII for 60 minutes at 35 degrees C. The correlation of the molecular species of the enzyme to CDA was determined by fractionating CI:CII mixtures over an anion exchange column. Individual fractions were analyzed for A280, CDA activity, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to correlate chromatographic analysis of these enzyme mixtures to the molecular species of collagenase effective in collagen degradation. RESULTS: CI has approximately 6 to 17 x higher specific activity than CII in this assay. Assays of different individual fractions recovered after anion exchange chromatography showed that the CDA of collagenase was dependent on the molecular species of the enzyme. Only intact CII and CI with molecular weights >or=100 kDa could degrade collagen fibrils. CONCLUSIONS: This assay provides a more reliable assessment of the functional activity of collagenase enzymes than peptide substrates currently used today. Fractionation of purified collagenase mixtures by anion exchange chromatography followed by analysis of individual fractions by SDS-PAGE and CDA assays will provide a powerful tool to analyze the molecular species of CI and CII required for islet isolation.

Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss).

Amyloid fibrils were isolated from cardiac tissue of two brothers who died from familial amyloidotic polyneuropathy (FAP) type II. Sequence analysis on peptides derived from proteolytic cleavage with trypsin and fragmentation with cyanogen bromide reveal that the fibril subunit protein is derived from plasma transthyretin (prealbumin). About two-thirds of the fibril subunit protein was found to contain an amino acid substitution at position 84 where the normal isoleucine residue has been replaced by serine. Sequence analysis of the plasma transthyretin (prealbumin) from the two brothers as well as two clinically diagnosed FAP type II family members and two of four children of affected individuals showed the presence of serine at position 84. The presence of this substitution also correlates with low serum levels of retinol-binding protein and thus transthyretin (prealbumin) position 84 may be involved with the interaction of these two proteins.

Identification of carriers of a variant plasma prealbumin (transthyretin) associated with familial amyloidotic polyneuropathy type I.

A method is described for detecting carriers of a variant plasma prealbumin that is associated with familial amyloidotic polyneuropathy (FAP) type I. It is based on the finding of an extra methionine in the variant prealbumin, at position 30 from the amino terminals. Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the variant prealbumin gives three peptides. The extra can then be detected in two ways: by HPLC using a reverse phase C18 column, and by sequential Edman degradation. Each method can detect as little as 1% variant prealbumin in isolated plasma prealbumin, and therefore, can identify carriers of the gene for the variant protein. Since FAP type I usually is not manifest until after the childbearing years, this method to identify carriers of the gene offers a new approach for genetic counseling of families with this disease. To date, kindreds with hereditary amyloidosis that could benefit from these studies include those with FAP type I of Swedish, Japanese, and Portuguese origins.

Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person.

Serum amyloid A protein (SAA) is a major acute-phase protein in humans and most other mammals. In addition, it is the serum precursor of the major protein constituent of reactive amyloid fibrils. Sequence analyses have identified a number of polymorphic forms of human SAA and amyloid A protein (AA), but the question of the number of genes encoding SAA in the human has not been addressed. In addition, there are insufficient data to predict whether one form of SAA predisposes to amyloid fibril formation. In the present study three separate SAA proteins have been isolated from the plasma of one individual and completely sequenced. While two of the SAA forms (SAA2 alpha and SAA2 beta) differ from each other only at position 71, they differ from the most abundant form (SAA1) at seven and eight other positions, respectively. Nucleotide sequencing of cDNAs from a liver library of this individual identified all three mRNs coding for these proteins and proved that: (a) the often-reported absence of arginine at the amino terminus of SAA proteins must result from proteolytic processing of the protein; (b) the polymorphism involving histidine and arginine at position 71 is present at the DNA level and therefore is not due to an event at the translational level; (c) there are at least two genes coding for human SAA. Comparison of these data to published sequences of SAA and AA proteins may help in identifying genetically determined forms of SAA which predispose to reactive amyloid fibril formation.

Biochemical and molecular genetic characterization of a new variant prealbumin associated with hereditary amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant late-onset disorder characterized by the extracellular deposition of amyloid fibrils. In all cases studied these fibrils have been found to be composed of plasma prealbumin (transthyretin) containing a single amino acid substitution. Biochemical studies were conducted on amyloid from one patient and plasma prealbumin from his affected brother, both part of a large kindred from the Appalachian region of the United States. Sequence analysis of the amyloid subunit protein showed it to be prealbumin with about two-thirds of the molecules containing a substitution of alanine for threonine at position 60. Studies of the plasma prealbumin showed that the same substitution was present in 40-45% of the protein. Based on this substitution and the prealbumin cDNA sequence, a Pvu II restriction fragment length DNA polymorphism (RFLP) was predicted and demonstrated in DNA of both patients as well as other family members. This RFLP confirms the predicted DNA mutation responsible for the protein variant, and represents an accurate method for detection of this gene.

Identification of a new hereditary amyloidosis prealbumin variant, Tyr-77, and detection of the gene by DNA analysis.

In the last several years, five human plasma prealbumin (transthyretin) variants have been discovered in association with hereditary amyloidosis, a late-onset fatal disorder. We recently studied a patient of German descent with peripheral neuropathy and bowel dysfunction. Biopsied rectal tissue contained amyloid that stained with anti-human prealbumin. Amino acid sequence analysis of the patient's plasma prealbumin revealed both normal and variant prealbumin molecules, with the variant containing a tyrosine at position 77 instead of serine. We predicted a single nucleotide change in codon 77 of the variant prealbumin gene, which we then detected in the patient's DNA using the restriction enzyme SspI and a specifically tailored genomic prealbumin probe. DNA tests of other family members identified several gene carriers. This is the sixth prealbumin variant implicated in amyloidosis, and adds to the accumulating evidence that the prealbumin amyloidoses are more varied and prevalent than previously thought.

Complete amino acid sequence of the major component myoglobin from Hubb's beaked whale, Mesoplodon carlhubbsi.

The complete primary structure of the major component myoglobin from Hubb's beaked whale, Mesoplodon carlhubbsi, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. In all experiments a Beckman 890C automatic sequencer was used to degrade the peptides. A cyanogen bromide digest was used to fragment the protein at its two methionine residues into three fragments which were separated by gel filtration. In a similar pattern the protein was citraconylated to protect the lysine residues and the modified protein fragmented at its arginine residues with trypsin. The three peptides obtained here were also purified by gel filtration. Sequencer analysis of the whole apoprotein, cyanogen bromide fragments and the peptides cleaved at the arginine residues with trypsin provided 80% of the completed sequence. The remainder of the sequence was obtained by digesting the middle cyanogen bromide fragment with staphylococcal protease and by total tryptic digestion of the whole apoprotein and isolating the resulting peptides by ion-exchange chromatography. The primary structure of the Mesoplodon carlhubbsi myoglobin represents a sequence which may be closey related to the ancestral sequence of all cetacean myoglobins.

Complete amino acid sequence of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris.

The complete primary structure of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at its two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage with trypsin of the citraconylated apomyoglobin at its three arginine residues. Further digestion of the central cyanogen bromide peptide with S. aureus strain V8 protease and the 1,2-cyclohexanedione-treated central cyanogen bromide peptide with trypsin enabled the determination of the remainder of the covalent structure. This myoglobin differs from the cetacean myoglobins determined to date at 12 to 17 positions. These large sequence differences reflect the distant taxonomic relationships between the goose-beaked whale and the other species of Cetacea the myoglobin sequences of which have previously been determined.

Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Amino acid sequence of a kappa I primary (AL) amyloid protein (AND).

The complete amino acid sequence of amyloid protein AND is presented. Amyloid fibrils were isolated from the spleen of patient AND, and the subunit protein was isolated from the fibrils after reduction and carboxymethylation. Sequence analysis of intact protein AND identified it as a kappa I immunoglobulin light chain. The complete sequence was determined from its tryptic peptides. The protein contained the entire variable region and the constant region to position 145 of the light chain. Several unique amino acid substitutions were found in protein AND compared to other kappa I proteins. The glycine, serine, arginine, threonine, alanine and arginine at positions 31, 45, 55, 76, 85 and 107, respectively, are reported for the first time in a kappa I protein. A number of uncommon amino acid substitutions were found in the framework regions in protein AND around the contact region of the dimer which may result in the molecule becoming more susceptible to fibril formation.

Polymorphism in a kappa I primary (AL) amyloid protein (BAN).

In an attempt to understand the relationship of amino acid sequence to the formation of primary or multiple myeloma-related amyloid (AL amyloid), we have determined the complete amino acid sequence of amyloid protein BAN. This protein belongs to the kappa I immunoglobulin light chain subgroup and has a polypeptide chain length of 126 amino acids. It encompasses the entire variable region, the joining segment and the first tryptic peptide of the constant region. This protein has two unique features. First, the molecule is glycosylated. At position 61 the usual arginine residue has been replaced by an asparagine with the generation of the signal sequence Asn-Phe-Thr, to which a glucosamine-containing carbohydrate unit is attached. Secondly, the protein is not monoclonal but consists of two chains which have the same variable region but different J-segments. Comparison of the BAN sequence with other amyloid and nonamyloid kappa I proteins reveals a systematic difference between the two groups. In the amyloid proteins, several hydrophilic framework residues have been replaced by hydrophobic residues. These substitutions may provide the nucleation sites for self-aggregation and fibril formation.

Reduced affinity for thyroxine in two of three structural thyroxine-binding prealbumin variants associated with familial amyloidotic polyneuropathy.

T4-binding prealbumin (TBPA), a protein synthesized by the liver, circulates as a tetramer and transports 15-20% of T4. We studied 3 variants of the TBPA monomer recently identified in serum and amyloid fibrils of patients affected by familial amyloidotic polyneuropathy (FAP). They represent single amino acid substitutions at positions 30 (type I), 60 (Appalachian), and 84 (type II). Tests of thyroid function and the apparent association constant (Ka) of T4 binding to TBPA were measured in whole serum from 14 carriers of FAP identified clinically, by amino acid sequence analysis, or by DNA restriction fragment analysis. Significant reduction of Ka was found in subjects with FAP types I and II, but not in subjects with the Appalachian type. Mean (+/- SD) values of 0.24 +/- 0.08 X 10(7) M-1 for type I and 0.26 +/- 0.10 X 10(7) M-1 for type II were significantly (P less than 0.0001) lower than those for normal relatives (1.39 +/- 0.30 X 10(7) M-1) or unrelated normal subjects (1.41 +/- 0.18 X 10(7) M-1). The mean Ka value for the five subjects with FAP of the Appalachian type was slightly but not significantly reduced (1.08 +/- 0.11 X 10(7) M-1). There was no overlap of individual Ka values of subjects with types I and II TBPA with those of subjects from all other groups. Abnormalities of thyroid function included slight but significant reductions of the mean total serum T4 concentration in the subjects with type II FAP and the mean serum total T3 concentration in those with type I FAP. Four subjects with FAP (two type II and two of the Appalachian type) had biochemical evidence of hypothyroidism. The three subjects with total serum T3 levels below the limit of normal had amyloid cardiomyopathy. These results indicate that TBPAs from subjects with FAP types I and II have relatively lower affinity for T4. Although none of the substituted amino acids in these variant TBPAs contribute directly to the surface of the putative T4-binding site, the side chains of amino acids 30 and 84, but not 60, interact with internal residues of the beta-structure which forms the presumed binding site, in agreement with our results of Ka measurements. The high incidence of hypothyroidism is due to the probably fortuitous occurrence of Hashimoto's thyroiditis as well as to partial destruction of the thyroid gland by amyloid deposits.

Characterization and functional assessment of Clostridium histolyticum class I (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase.

OBJECTIVES: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. METHODS: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. RESULTS: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. CONCLUSIONS: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.

Tissue dissociation enzyme neutral protease assessment.

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.

Primary structure of amyloid fibril protein AA in azocasein-induced amyloidosis of CBA/J mice.

Secondary amyloidosis was induced in CBA/J mice by subcutaneous injections of azocasein after priming with amyloid-enhancing factor. Amyloid fibrils were isolated from spleens and the subunit amyloid A (AA) protein purified by gel filtration on a column of Sepharose CL6B. The AA protein was fragmented with trypsin, cyanogen bromide, and Staphylococcus protease, and the peptides were purified by reverse-phase high-performance liquid chromatography. This protein is composed of 73 amino acid residues arranged in a single polypeptide chain with homogeneous amino and carboxyl terminals. Sequence homology with protein AA from other species is quite high with near identity for residues 31 through 54. This sequence is identical to the partial structures for CBA/J mouse AA and serum amyloid A (SAA) previously reported. It is also an exact match to a predicted 73-residue segment from one form of mouse SAA complementary DNA.

Prealbumin and retinol binding protein serum concentrations in the Indiana type hereditary amyloidosis.

Serum prealbumin and retinol binding protein (RBP) concentrations were determined for 68 members of a kindred in Indiana with a familial type of systemic amyloidosis. Immunohistochemical studies on rectal and muscle biopsy material from individuals with this type of amyloidosis revealed staining of amyloid deposits with anti-prealbumin. Both the serum prealbumin and RBP concentrations were significantly depressed in 9 patients with amyloidosis when compared with normal controls and unaffected kin. In addition, the mean RBP serum concentration of 21 offspring of the patients with amyloidosis was significantly depressed. A more significant finding was that on the basis of the serum RBP concentrations, the offspring could be divided into 2 distinct groups. One group represented approximately 50% of the children and had serum prealbumin and RBP concentrations not significantly different from their afflicted parents. The second group had serum prealbumin and RBP concentrations not significantly different from those of normal controls and non-affected kin. These findings show that prealbumin and RBP serum concentrations are depressed in patients with the Indiana type of hereditary amyloidosis and that these serum abnormalities may be present long before development of clinical disease. They suggest that individuals with this genetic abnormality may be identified prior to clinical expression of the disease.

Polymorphism of human plasma thyroxine binding prealbumin.

Amyloid fibrils from an individual with heredofamilial amyloidosis were found to be composed of plasma prealbumin. To study this protein a three step procedure to isolate prealbumin from plasma was developed. It entailed ion exchange chromatography on DEAE Sephadex, affinity chromatography on Affi-gel Blue and gel filtration on AcA-34. Trypsin Digests of prealbumin were separated by reverse phase HPLC and the pattern compared to that from the normal protein. Only one unexpected peptide was found and it represented the substitution of a methionine for a valine at position 30 in the molecule. This substitution accounts for about 1/3 of the isolated molecules and it represents the first point mutation identified in human plasma prealbumin.