International collaborative study to assess new stocks of candidate reference preparations to control the level of anti-D in IVIG

The level of anti-D antibodies in human immunoglobulin products for intravenous administration (IVIG) is controlled by the direct haemagglutination method prescribed by the European Pharmacopoeia (Ph. Eur.) that requires 2 control reference reagents. The World Health Organization (WHO) positive control International Reference Reagent (IRR; 02/228) with a nominal titre of 8 defines the highest acceptable titre, while the negative control preparation (02/226) has a nominal titre of <2. Working reference preparations (04/132 and 04/140) were subsequently established as Biological Reference Preparations (BRPs) for the Ph. Eur., and for distribution by the United States Food and Drug Administration (US FDA) and the National Institute for Biological Standards and Control (NIBSC). Due to diminishing stocks of these working reference preparations across the 3 institutions, a joint international study was organised to establish harmonised replacement batches. Sixteen laboratories contributed data to the study to evaluate positive and negative candidate replacement batches (13/148 and 12/300, respectively) against the WHO positive and negative control IRRs and the current working reference preparations (BRPs). The results show that the candidate reference preparations (13/148 and 12/300) are indistinguishable from the corresponding IRRs and current BRPs. The candidate preparations 13/148 and 12/300 were adopted by the Ph. Eur. Commission as Immunoglobulin (anti-D antibodies test) BRP batch 2 and Immunoglobulin (anti-D antibodies test negative control) BRP batch 2 with nominal haemagglutination titres of 8 and <2, respectively. The same materials were also adopted as NIBSC and US FDA reference preparations, thus ensuring full harmonisation.

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.

International collaborative study to establish immunoglobulin (anti-D test) BRP batch 1.

An international collaborative study was organised to establish a European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) and United States (US) Food and Drug Administration (FDA) reference preparation for the test for anti-D (anti-Rho) antibodies in human normal immunoglobulin for intravenous administration (IGIV). A candidate positive control (IGIV+anti-D) and negative control IGIV were compared to corresponding World Health Organization (WHO) International Reference Reagents using a direct haemagglutination reference method. Sixteen (16) laboratories participated in the collaborative study. Further to completion of the study, the materials assayed in the study were granted the status of Ph. Eur. and US FDA reference preparations for controlling the levels of anti-D in IGIV.

Structure and function of desmosomal transmembrane core and plaque molecules.

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.

Structure of desmoplakin and its association with intermediate filaments.

Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)

International collaborative study to evaluate a candidate reference preparation to define an appropriate specified limit of anti-D in intravenous immunoglobulin products.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate a lyophilized intravenous immunoglobulin (IVIG) preparation containing anti-D (02/228; nominal reciprocal titre of 8) for its suitability to define the maximum limit of anti-D in IVIG products when used in a proposed reference method of direct haemagglutination of papain-treated erythrocytes, in an international collaborative study. MATERIALS AND METHODS: Twenty laboratories tested 02/228 along with a negative control IVIG preparation and four IVIG samples containing different levels of anti-D. Nineteen laboratories performed direct haemagglutination methodology using papain-treated erythrocytes; five of these laboratories and one additional laboratory performed their in-house haemagglutination methodology (all indirect antiglobulin tests). RESULTS: The mode titre of 02/228, obtained by using the proposed reference method, was 8 (62.5% of tests). However, there was wide variation in haemagglutination titres between laboratories for three of the four samples. Correcting the titres of the samples relative to those of the proposed reference preparation reduced the interlaboratory variability and increased the frequency of the mode titres in three out of four samples. The indirect antiglobulin tests also showed wide interlaboratory variability and were less sensitive than the direct method in four laboratories. Eleven of the 14 laboratories that expressed an opinion considered that the level of anti-D in 02/228 was appropriate to define a specified limit. CONCLUSIONS: Our results demonstrate the necessity of using a reference preparation to define the maximum level of anti-D in IVIG products and ensure sufficient sensitivity in haemagglutination testing methodology. On the basis of these results, members of the European Pharmacopoeia Expert Group 6B recommended revision of the appropriate monograph to include this new specification and test. The Food and Drug Administration in the USA intends to adopt the same maximal specification defined by the reference preparation and to recommend the same test for the safety of IVIG products. Preparations 02/228 and 02/226 were also established by the World Health Organization as International Reference Reagents to standardize haemagglutination testing for anti-D in normal IVIG products.

Molecular structure of the human desmoplakin I and II amino terminus.

Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.

Structure of the human desmoplakins. Implications for function in the desmosomal plaque.

Desmoplakins (DPs) I and II are two major related proteins located in the innermost portion of the desmosomal plaque where it is thought they may play a role in attaching intermediate filaments (IF) to the cell surface. We have isolated and sequenced human cDNA clones encoding two major DP domains and a portion of a third. These clones can be divided into two classes that we believe to represent DPI and DPII cDNAs; our evidence suggests that the DPII message is derived at least in part from the processing of a larger transcript encoded by a single gene. Computer-aided analysis of the DPI-predicted amino acid sequence indicates that the central domain, which contains the heptad repeat characteristic of many alpha-fibrous proteins, will participate in the formation of a coiled coil dimer approximately 130 nm in length. The periodicity of acidic and basic residues in the rod suggests that DPI will aggregate with itself or similar molecules into higher order filamentous structures. The carboxyl terminus contains three regions with significant homology, each of which comprises almost five repeats of a 38-residue motif. It is likely that these regions each fold into a compact globular conformation stabilized by intrachain ionic interactions. Comparison of the predicted amino acid sequence of a cDNA encoding a portion of the 230-kDa bullous pemphigoid antigen (Stanley, J. R., Tanaka, T., Mueller, S., Klaus-Kovtun, V., and Roop, D. (1988) J. Clin. Invest. 82, 1864-1870) with DP revealed the presence of a 38-residue repeat with striking similarity to that of the DPs. Significantly, the periodicity in acidic and basic residues of these domains is the same as that found in the 1B rod domain of IF proteins. This suggests the possibility that the DPs might interact with IF via their common periodicity of charged residues.