Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (alphabeta)2 heterotetramer of two non‐identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three‐dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N‐terminal residue of the beta subunit and most importantly its alpha‐amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three‐dimensional structure and whose catalytic mechanisms with an N‐terminal nucleophile seem basically to be similar.

Congenital deficiency in coagulation factor XIII is a rare autosomal recessive bleeding disorder. Although the defect was characterized over 30 years ago, little is known about the molecular basis of the disorder. Here, we show two novel point mutations in the gene of the A- subunit of factor XIII in the genetically isolated population of Finland. All eight factor XIII-deficient families identified in Finland were studied. The exons of the gene of A-subunit were amplified individually by polymerase chain reaction and subsequently screened by single-strand conformation polymorphism. Sequence analysis of the abnormally migrating fragments showed two point mutations resulting in an amino acid alteration. A C-to-T transition at Arg-661 in exon XIV created a premature stop codon. This mutation was detected in six of the eight families, thus being the major alteration causing FXIII deficiency in Finland. In two of the six families, the patients were compound heterozygotes with the Arg-661-Stop mutation in one allele and either a T-to-C point mutation in exon VI or a thus far uncharacterized mutation in the other allele. The T-to-C transition in exon VI resulted in a substitution of threonine for methionine 242. The transition was found in one family only, where it was in the heterozygote form combined with the Arg-661-Stop mutation. To evaluate the consequences of these mutations, steady-state FXIII mRNA levels were quantitated by solid-phase minisequencing. In addition to the termination of translation 70 amino acids before the initial stop codon, the Arg-661- Stop mutation causes a 10- to 30-fold reduction in FXIII mRNA levels. This is also likely to result in a low translation level in the truncated polypeptide. In contrast, Met-242-Thr mutation does not seem to affect the level of mRNA. Here, the absence of a functional and immunodetectable protein is probably caused by an altered conformation of the mutant polypeptide, resulting in early degradation of the defective protein.

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The thermoregulatory function of the skin differs in adult cold‐acclimated and heat‐acclimated rock pigeons (Columba livia). In general, the cutaneous evaporative cooling mechanism is not activated by appropriate stimuli in cold‐acclimated pigeons in contrast to heat‐acclimated pigeons. We studied with electron microscopy whether the differences in the function of the skin are reflected in the structure of the epidermal water barrier of these two extreme acclimation states. The epidermis of cold‐acclimated pigeons is attenuated, and the underlying dermis lacks any intimate vascularization. Both the extracellular and the intracellular domains in the stratum corneum contain organized lamellar lipids. At the stratum transitivum–stratum corneum interface, multigranular body secretion is indicated by the highly convoluted cell membranes and membraneous sacculae enclosing the multigranular bodies. Alternatively, multigranular bodies retain in the corneocytes, and the lipoid material originated from them is reprocessed to broad lamellae. The keratohyalin (KH) granules are spotlike and oriented as cortical bands beneath the plasma membrane. In heat‐acclimated pigeons, the epidermis displays modified patches side by side with basic structural type of epidermis. The modified areas are characterized by hypertrophy and abundance of dermal capillaries adjacent to the hypertrophied patch. No lamellar lipids are discerned in the dilated extracellular space. The structure of multigranular bodies is abnormal, and the numbers of lipid droplets in the outer viable epidermis and stratum corneum are decreased. The transitional cells contain stellate KH granules, which form a network throughout the cell. It is concluded that cold‐acclimated pigeons have a lamellar, extracellular water barrier, the cutaneous water evaporation is minimized, and heat is stored in the body core. Acclimation to heat leads to formation of structurally heterogeneous skin. The structurally modified skin patches show disruption of the barrier‐forming machinery in the multigranular bodies and marked reorganization of fibrillar proteins and electron‐dense KH masses in the transitional layer. Thus water barrier adjustments in cold‐ and heat‐acclimated pigeons manifest the dynamic function of avian skin as a thermoregulatory organ. J. Morphol. 246:118–130, 2000 © 2000 Wiley‐Liss, Inc.